The role of NF-κB in the expression of inflammatory genes and its participation in the overall inflammatory process of chronic diseases and acute tissue injury are well established. We and others have demonstrated a critical involvement of poly(ADP-ribose) polymerase (PARP)-1 during inflammation, in part, through its relationship with NF-κB. However, the mechanism by which PARP-1 affects NF-κB activation has been elusive. In this study, we show that PARP-1 inhibition by gene knockout, knockdown, or pharmacologic blockade prevented p65 NF-κB nuclear translocation in smooth muscle cells upon TLR4 stimulation, NF-κB DNA-binding activity, and subsequent inducible NO synthase and ICAM-1 expression. Such defects were reversed by reconstitution of PARP-1 expression. PARP-1 was dispensable for LPS-induced IκBα phosphorylation and subsequent degradation but was required for p65 NF-κB phosphorylation. A perinuclear p65 NF-κB localization in LPS-treated PARP-1^−/− cells was associated with an export rather an import defect. Indeed, whereas PARP-1 deficiency did not alter expression of importin α3 and importin α4 and their cytosolic localization, the cytosolic levels of exportin (Crm)-1 were increased. Crm1 inhibition promoted p65 NF-κB nuclear accumulation as well as reversed LPS-induced p65 NF-κB phosphorylation and inducible NO synthase and ICAM-1 expression. Interestingly, p65 NF-κB poly(ADP-ribosyl)ation decreased its interaction with Crm1 in vitro. Pharmacologic inhibition of PARP-1 increased p65 NF-κB–Crm1 interaction in LPS-treated smooth muscle cells. These results suggest that p65 NF-κB poly(ADP-ribosyl)ation may be a critical determinant for the interaction with Crm1 and its nuclear retention upon TLR4 stimulation. These results provide novel insights into the mechanism by which PARP-1 promotes NF-κB nuclear retention, which ultimately can influence NF-κB–dependent gene regulation.

Airway inflammation is a central feature of asthma and chronic obstructive pulmonary disease. Reactive oxygen species (ROS) contribute to inflammation by damaging DNA, which, in turn, results in the activation of poly(ADP-ribose) polymerase-1 (PARP-1) and depletion of its substrate, nicotinamide adenine dinucleotide. Here we show that prevention of PARP-1 activation protects against both ROS-induced airway epithelial cell injury in vitro and airway inflammation in vivo. H2O2 induced the generation of ROS, PARP-1 activation and concomitant nicotinamide adenine dinucleotide depletion, and release of lactate dehydrogenase in A549 human airway epithelial cells. These effects were blocked by the PARP-1 inhibitor 3-aminobenzamide (3-AB). Furthermore, 3-AB inhibited both activation of the proinflammatory transcription factor nuclear factor-κB and expression of the interleukin-8 gene induced by H2O2 in these cells. In a murine model of allergen-induced asthma, 3-AB prevented airway inflammation elicited by ovalbumin. Moreover, PARP-1 knockout mice were resistant to such ovalbumin-induced inflammation. These protective effects were associated with an inhibition of expression of the inducible nitric oxide synthase. These results implicate PARP-1 activation in airway inflammation, and suggest this enzyme as a potential target for the development of new therapeutic strategies in the treatment of asthma as well as other respiratory disorders such as chronic obstructive pulmonary disease.

Background: Poly(ADP-ribose) polymerase (PARP) inhibitors (eg, olaparib) are effective against BRCA-mutated cancers at/near maximum tolerated doses by trapping PARP-1 on damaged chromatin, benefitting only small patient proportions. The benefits of targeting non-DNA repair aspects of PARP with metronomic doses remain unexplored. Methods: Colon epithelial cells or mouse or human bone marrow (BM)-derived-myeloid-derived suppressor cells (MDSCs) were stimulated to assess the effect of partial PARP-1 inhibition on inflammatory gene expression or immune suppression. Mice treated with azoxymethane/four dextran-sulfate-sodium cycles or APC^Min/+ mice bred into PARP-1^+/− or treated with olaparib were used to examine the role of PARP-1 in colitis-induced or spontaneous colon cancer, respectively. Syngeneic MC-38 cell-based (microsatellite instability, MSI^high) or CT-26 cell-based (microsatellite stable, MSS) tumor models were used to assess the effects of PARP inhibition on host responses and synergy with anti-Programmed cell Death protein (PD)-1 immunotherapy. Results: Partial PARP-1 inhibition, via gene heterozygosity or a moderate dose of olaparib, protected against colitis-mediated/APC^Min -mediated intestinal tumorigenesis and APC^Min -associated cachexia, while extensive inhibition, via gene knockout or a high dose of olaparib, was ineffective or aggravating. A sub-IC50-olaparib dose or PARP-1 heterozygosity was sufficient to block tumorigenesis in a syngeneic colon cancer model by modulating the suppressive function, but not intratumoral migration or differentiation, of MDSCs, with concomitant increases in intratumoral T cell function and cytotoxicity, as assessed by granzyme-B/interferon-γ levels. Adoptive transfer of WT-BM-MDSCs abolished the protective effects of PARP-1 heterozygosity. The mechanism of MDSC modulation involved a reduction in arginase-1/inducible nitric oxide synthase/cyclo-oxygenase-2, but independent of PARP-1 trapping on chromatin. Although a high-concentration olaparib or the high-trapping PARP inhibitor, talazoparib, activated stimulator of interferon gene (STING) in BRCA-proficient cells and induced DNA damage, sub-IC50 concentrations of either drug failed to induce activation of the dsDNA break sensor. STING expression appeared dispensable for MDSC suppressive function and was not strictly required for olaparib-mediated effects. Ironically, STING activation blocked human and mouse MDSC function with no additive effects with olaparib. A metronomic dose of olaparib was highly synergistic with anti-PD-1-based immunotherapy, leading to eradication of MSI^high or reduction of MSS tumors in mice. Conclusions: These results support a paradigm-shifting concept that expands the utility of PARP inhibitor and encourage testing metronomic dosing of PARP inhibitor to enhance the efficacy of checkpoint inhibitor-based immunotherapies in cancer.

Although a relationship between PDZK1 expression and estrogen receptor (ER)-α stimulation has been suggested, the nature of such a connection and the function of PDZK1 in breast cancer remain unknown. Human tissue microarrays (cancer tissue: 262 cores; normal tissue: 87 cores) and breast cancer cell lines were used to conduct the study. We show that PDZK1 protein expression is tightly correlated with human breast malignancy, is negatively correlated with age and had no significant correlation with ER-α expression levels. PDZK1 exhibited an exclusive epithelial expression with mostly cytosolic subcellular localization. Additionally, 17β-estradiol induced PDZK1 expression above its basal level more than 24 h after treatment in MCF-7 cells. PDZK1 expression was indirectly regulated by ER-α stimulation, requiring insulinlike growth factor 1 receptor (IGF-1R) expression and function. The molecular link between PDZK1 and IGF-1R was supported by a significant correlation between protein and mRNA levels (r = 0.591, p < 0.001, and r = 0.537, p < 0.001, respectively) of the two factors in two different cohorts of human breast cancer tissues. Interestingly, PDZK1 knockdown in MCF-7 cells blocked ER-dependent growth and reduced c-Myc expression, whereas ectopic expression of PDZK1 enhanced cell proliferation in the presence or absence of 17β-estradiol potentially through an increase in c-Myc expression, suggesting that PDZK1 has oncogenic activity. PDKZ1 also appeared to interact with the Src/ER-α/epidermal growth factor receptor (EGFR) complex, but not with IGF-1R and enhanced EGFR-stimulated MEK/ERK1/2 signaling. Collectively, our results clarify the relationship between ER-α and PDZK1, propose a direct relationship between PDZK1 and IGF-1R, and identify a novel oncogenic activity for PDZK1 in breast cancer.

Caspase-activated DNase (CAD) is the most favorable candidate for chromatin degradation during apoptosis. Ca²⁺-dependent endonucleases are equally important in internucleosomal DNA fragmentation (INDF), including the PARP-1-regulated DNAS1L3. Despite the elaborate work on these endonucleases, the question of whether these enzymes cooperate during INDF was not addressed. Here, we show a lack of correlation between INDF and CAD expression levels and inactivation by cleavage of its inhibitor (ICAD) during apoptosis. The cells that failed to induce INDF accumulated large amounts of 50-kb breaks, which is suggestive of incomplete chromatin processing. Similarly, INDF was blocked by Ca²⁺ chelation without a block in ICAD cleavage or caspase-3 activation, which is consistent with the involvement of CAD in 50-kb DNA fragmentation and its Ca²⁺ independence. However, DNAS1L3 expression in INDF-deficient cells promoted INDF during apoptosis and was blocked by Ca²⁺ chelation. Interestingly, expression of DNAS1L3 in ICAD-deficient cells failed to promote tumor necrosis factor α-induced INDF but required the coexpression of ICAD. These results suggest a cooperative activity between CAD and DNAS1L3 to accomplish INDF. In HT-29 cells, endogenous DNAS1L3 localized to the endoplasmic reticulum (ER) and translocated to the nucleus upon apoptosis induction but prior to INDF manifestation, making it the first reported Ca²⁺-dependent endonuclease to migrate from the ER to the nucleus. The nuclear accumulation of DNAS1L3, but not its exit out of the ER, required the activity of cysteine and serine proteases. Interestingly, the endonuclease accumulated in the cytosol upon inhibition of serine, but not cysteine, proteases. These results exemplify the complexity of chromatin degradation during apoptosis.