Differential Localisation of PARP-1 N-Terminal Fragment in PARP-1+/+ and PARP-1?/? Murine Cells

Human PARP family consists of 17 members of which PARP-1 is a prominent member and plays a key role in DNA repair pathways. It has an N-terminal DNA-binding domain (DBD) encompassing the nuclear localisation signal (NLS), central automodification domain and C-terminal catalytic domain. PARP-1 accounts for majority of poly-(ADP-ribose) polymer synthesis that upon binding to numerous proteins including PARP itself modulates their activity. Reduced PARP-1 activity in ageing human samples and its deficiency leading to telomere shortening has been reported. Hence for cell survival, maintenance of genomic integrity and longevity presence of intact PARP-1 in the nucleus is paramount. Although localisation of full-length and truncated PARP-1 in PARP-1 proficient cells is well documented, subcellular distribution of PARP-1 fragments in the absence of endogenous PARP-1 is not known. Here we report the differential localisation of PARP-1 N-terminal fragment encompassing NLS in PARP-1^+/+ and PARP-1^−/− mouse embryo fibroblasts by live imaging of cells transiently expressing EGFP tagged fragment. In PARP-1^+/+ cells the fragment localises to the nuclei presenting a granular pattern. Furthermore, it is densely packaged in the midsections of the nucleus. In contrast, the fragment localises exclusively to the cytoplasm in PARP-1^−/− cells. Flourescence intensity analysis further confirmed this observation indicating that the N-terminal fragment requires endogenous PARP-1 for its nuclear transport. Our study illustrates the trafficking role of PARP-1 independently of its enzymatic activity and highlights the possibility that full-length PARP-1 may play a key role in the nuclear transport of its siblings and other molecules.     

PARP-1 inhibition does not restore oxidant-mediated reduction in SIRT1 activity

Sirtuin1 (SIRT1) deacetylase and poly(ADP-ribose)-polymerase-1 (PARP-1) respond to environmental cues, and both require NAD⁺ cofactor for their enzymatic activities. However, the functional link between environmental/oxidative stress-mediated activation of PARP-1 and SIRT1 through NAD⁺ cofactor availability is not known. We investigated whether NAD⁺ depletion by PARP-1 activation plays a role in environmental stimuli/oxidant-induced reduction in SIRT1 activity. Both H₂O₂ and cigarette smoke (CS) decreased intracellular NAD⁺ levels in vitro in lung epithelial cells and in vivo in lungs of mice exposed to CS. Pharmacological PARP-1 inhibition prevented oxidant-induced NAD⁺ loss and attenuated loss of SIRT1 activity. Oxidants decreased SIRT1 activity in lung epithelial cells; however increasing cellular NAD⁺ cofactor levels by PARP-1 inhibition or NAD⁺ precursors was unable to restore SIRT1 activity. SIRT1 was found to be carbonylated by CS, which was not reversed by PARP-1 inhibition or selective SIRT1 activator. Overall, these data suggest that environmental/oxidant stress-induced SIRT1 down-regulation and PARP-1 activation are independent events despite both enzymes sharing the same cofactor.     

Irgm 1参与动脉粥样硬化斑块的形成

目的:探讨免疫相关GTP酶1(Irgm 1)对小鼠血管动脉粥样硬化(AS)斑块形成的影响。方法:高脂饲料喂养野生型(WT)、ApoE~(-/-)Irgm 1~(+/+)和ApoE~(-/-)Irgm1~(+/-)小鼠3个月,建立AS模型;取小鼠主动脉弓,免疫荧光染色方法观察WT和ApoE~(-/-)Irgm 1~(+/+)小鼠血管AS斑块中Irgm 1的表达情况及部位;Western blot方法检测WT和ApoE~(-/-)Irgm 1~(+/+)小鼠血管AS斑块中Irgm 1蛋白表达情况;Q-PCR方法检测WT和ApoE~(-/-)Irgm 1~(+/+)小鼠血管AS斑块中Irgm 1 m RNA表达情况;油红O染色观察ApoE~(-/-)Irgm1~(+/+)和ApoE~(-/-)Irgm1~(+/-)小鼠血管AS斑块形成情况;结果:与WT组相比,ApoE~(-/-)Irgm 1~(+/+)组小鼠主动脉弓AS斑块中Irgm 1+细胞明显增多,Irgm 1+细胞主要位于血管AS斑块的表面;与WT组相比,ApoE~(-/-)Irgm 1~(+/+)组小鼠血管AS斑块中Irgm 1蛋白表达显著增多(P0.001),Irgm 1 m RNA表达显著增多(P0.01);与ApoE~(-/-)Irgm1~(+/-)组相比,ApoE~(-/-)Irgm1~(+/+)组小鼠主动脉弓AS斑块面积显著增大(P0.01);结论:Irgm 1能够促进血管AS斑块的形成。     

TLR2 mediates immunity to experimental cysticercosis

Information concerning TLR-mediated antigen recognition and regulation of immune responses during helminth infections is scarce. TLR2 is a key molecule required for innate immunity and is involved in the recognition of a wide range of viruses, bacteria, fungi and parasites. Here, we evaluated the role of TLR2 in a Taenia crassiceps cysticercosis model. We compared the course of T. crassiceps infection in C57BL/6 TLR2 knockout mice (TLR2^-/-) with that in wild type C57BL/6 (TLR2^+/+) mice. In addition, we assessed serum antibody and cytokine profiles, splenic cellular responses and cytokine profiles and the recruitment of alternatively activated macrophages (AAMφs) to the site of the infection. Unlike wild type mice, TLR2^-/- mice failed to produce significant levels of inflammatory cytokines in either the serum or the spleen during the first two weeks of Taenia infection. TLR2^-/- mice developed a Th2-dominant immune response, whereas TLR2^+/+ mice developed a Th1-dominant immune response after Taenia infection. The insufficient production of inflammatory cytokines at early time points and the lack of Th1-dominant adaptive immunity in TLR2^-/- mice were associated with significantly elevated parasite burdens; in contrast, TLR2^+/+ mice were resistant to infection. Furthermore, increased recruitment of AAMφs expressing PD-L1, PD-L2, OX40L and mannose receptor was observed in TLR2^-/- mice. Collectively, these findings indicate that TLR2-dependent signaling pathways are involved in the recognition of T. crassiceps and in the subsequent activation of the innate immune system and production of inflammatory cytokines, which appear to be essential to limit infection during experimental cysticercosis.     

Matrix Metalloproteinase-2 (MMP-2) Gene Deletion Enhances MMP-9 Activity,Impairs PARP-1 Degradation,and Exacerbates Hepatic Ischemia and Reperfusion Injury in Mice

Hepatic ischemia and reperfusion injury (IRI) is an inflammatory condition and a significant cause of morbidity and mortality after surgery. Matrix metalloproteinases (MMPs) have been widely implicated in the pathogenesis of inflammatory diseases. Among the different MMPs, gelatinases (MMP-2 and MMP-9) are within the most prominent MMPs detected during liver IRI. While the role of MMP-9 in liver damage has been fairly documented, direct evidence of the role for MMP-2 activity in hepatic IRI remains to be established. Due to the lack of suitable inhibitors to target individual MMPs in vivo, gene manipulation is as an essential tool to assess MMP direct contribution to liver injury. Hence, we used MMP-2^-/- deficient mice and MMP-2^+/+ wild-type littermates to examine the function of MMP-2 activity in hepatic IRI. MMP-2 expression was detected along the sinusoids of wild-type livers before and after surgery and in a small population of leukocytes post-IRI. Compared to MMP-2^+/+ mice, MMP-2 null (MMP-2^-/-) mice showed exacerbated liver damage at 6, 24, and 48 hours post-reperfusion, which was fatal in some cases. MMP-2 deficiency resulted in upregulation of MMP-9 activity, spontaneous leukocyte infiltration in naïve livers, and amplified MMP-9-dependent transmigration of leukocytes in vitro and after hepatic IRI. Moreover, complete loss of MMP-2 activity impaired the degradation of poly (ADP-ribose) polymerase (PARP-1) in extensively damaged livers post-reperfusion. However, the administration of a PARP-1 inhibitor to MMP-2 null mice restored liver preservation to almost comparable levels of MMP-2^+/+ mice post-IRI. Deficient PARP-1 degradation in MMP-2-null sinusoidal endothelial cells correlated with their increased cytotoxicity, evaluated by the measurement of LDH efflux in the medium. In conclusion, our results show for the first time that MMP-2 gene deletion exacerbates liver IRI. Moreover, they offer new insights into the MMP-2 modulation of inflammatory responses, which could be relevant for the design of new pharmacological MMP-targeted agents to treat hepatic IRI.     

TRAIL induces necroptosis involving RIPK1/RIPK3-dependent PARP-1 activation

Although TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand) is a well-known apoptosis inducer, we have previously demonstrated that acidic extracellular pH (pHe) switches TRAIL-induced apoptosis to regulated necrosis (or necroptosis) in human HT29 colon and HepG2 liver cancer cells. Here, we investigated the role of RIPK1 (receptor interacting protein kinase 1), RIPK3 and PARP-1 (poly (ADP-ribose) polymerase-1) in TRAIL-induced necroptosis in vitro and in concanavalin A (Con A)-induced murine hepatitis. Pretreatment of HT29 or HepG2 with pharmacological inhibitors of RIPK1 or PARP-1 (Nec-1 or PJ-34, respectively), or transient transfection with siRNAs against RIPK1 or RIPK3, inhibited both TRAIL-induced necroptosis and PARP-1-dependent intracellular ATP depletion demonstrating that RIPK1 and RIPK3 were involved upstream of PARP-1 activation and ATP depletion. In the mouse model of Con A-induced hepatitis, where death of mouse hepatocytes is dependent on TRAIL and NKT (Natural Killer T) cells, PARP-1 activity was positively correlated with liver injury and hepatitis was prevented both by Nec-1 or PJ-34. These data provide new insights into TRAIL-induced necroptosis with PARP-1 being active effector downstream of RIPK1/RIPK3 initiators and suggest that pharmacological inhibitors of RIPKs and PARP-1 could be new treatment options for immune-mediated hepatitis.     

The recently suggested intestinal cancer stem cell marker DCLK1 is an epigenetic biomarker for colorectal cancer

Recently, Dclk1 expression was identified to be an intestinal cancer stem cell specific biomarker in mouse models, implicating a potential role for targeting the DCLK1-postive cancer cells as a treatment for colorectal cancer. Using quantitative methylation specific PCR (qMSP) we here demonstrated that the DCLK1 promoter is hypermethylated in the vast majority of colorectal cancers (134/164; 82%), with no methylation in the normal mucosa samples (0/106). We further showed by Affymetrix exon arrays that DCLK1 is significantly downregulated in human colorectal cancer (n = 125) compared with normal colonic mucosa (n = 15), which was further confirmed by real-time RT-PCR of a subgroup of the samples. Additionally, a significant negative correlation was observed between methylation and DCLK1 expression in 74 cancer cell lines derived from 15 different tissues, and gene expression increased significantly after epigenetic drug treatment of initially methylated cancer cell lines. These findings underscore the potential of DCLK1 as a colorectal cancer biomarker for early detection, but may also have clinical implications regarding the previously proposed therapy toward DCLK1-positive cancer cells. This therapy would at best affect the cancer stem cell population, but will, based on the present results, not be efficient to treat the bulk of the tumor.     

Repair pathway for PARP-1 DNA-protein crosslinks

Poly(ADP-ribose) polymerase-1 (PARP-1) is a regulatory enzyme involved in many different processes of DNA and RNA metabolism, including DNA repair. Previously, PARP-1 was found capable of forming a covalent DNA-protein crosslink (DPC) at the apurinic/apyrimidinic (AP) site in double-stranded DNA. The C1´ atom of the AP site participates in Schiff base formation with a lysine side chain in PARP-1, and a covalent bond is formed upon reduction of the Schiff base. The PARP-1 DPC is formed in vivo where DPC formation correlates with AP site induction by a monofunctional alkylating agent. Here, we examined repair of PARP-1 DPCs in mouse fibroblasts and found that a proteasome inhibitor, MG-132, reduces repair resulting in accumulation of PARP-1 DPCs and increased alkylating agent cytotoxicity. Using a model DNA substrate mimicking the PARP-1 DPC after proteasomal degradation, we found that repair is completed by a sub-pathway of base excision repair (BER). Tyrosyl-DNA phosphodiesterase 1 was proficient in removing the ring-open AP site sugar at the phosphodiester linkage, leaving an intermediate for processing by other BER enzymes. The results reveal proteasomal degradation of the PARP-1 DPC is active in mouse fibroblasts and that a model repair intermediate is processed by the BER machinery.