Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.     

Haploinsufficiency of poly(ADP-ribose) polymerase-1-mediated poly(ADP-ribosyl)ation for centrosome duplication

The centrosome plays a vital role in maintaining chromosomal stability. Known as the microtubule organizing center, the centrosome is involved in the formation of spindle poles during mitosis, which ensures the distribution of the correct number of chromosomes to daughter cells. Aberrant centrosome duplication could cause centrosome amplification and chromosomal instability. We have previously shown that poly(ADP-ribose) polymerase-1 (PARP-1) is important for centrosome function and chromosomal stability. In this study, we used PARP-1(+/+), PARP-1(+/-) and PARP-1(-/-) primary mouse embryonic fibroblasts and found that the level of PARP-1 gene dosage correlates with PARP activity and the in vivo level of poly(ADP-ribosyl)ation, which could explain the mechanism by which PARP-1 haploinsufficiency affects centrosome duplication and chromosomal stability. Our results emphasize that correct regulation of poly(ADP-ribosyl)ation levels in vivo is important for maintenance of proper centrosome duplication and chromosomal stability.     

DNA maintenance following bleomycin-induced strand breaks does not require poly(ADP-ribosyl)ation activation in Drosophila S2 cells

BackgroundPoly-ADP ribosylation (PARylation) is a post translational modification, catalyzed by Poly(ADP-ribose)polymerase (PARP) family. In Drosophila, PARP-I (human PARP-1 ortholog) is considered to be the only enzymatically active isoform. PARylation is involved in various cellular processes such as DNA repair in case of base excision and strand-breaks.ObservationsStrand-breaks (SSB and DSB) are detrimental to cell viability and, in Drosophila, that has a unique PARP family organization, little is known on PARP involvement in the control of strand-breaks repair process. In our study, strands-breaks (SSB and DSB) are chemically induced in S2 Drosophila cells using bleomycin. These breaks are efficiently repaired in S2 cells. During the bleomycin treatment, changes in PARylation levels are only detectable in a few cells, and an increase in PARP-I and PARP-II mRNAs is only observed during the recovery period. These results differ strongly from those obtained with Human cells, where PARylation is strongly activating when DNA breaks are generated. Finally, in PARP knock-down cells, DNA stability is altered but no change in strand-breaks repair can be observed.ConclusionsPARP responses in DNA strands-breaks context are functional in Drosophila model as demonstrated by PARP-I and PARP-II mRNA increases. However, no modification of the global PARylation profile is observed during strand-breaks generation, only changes at cellular levels are detectable. Taking together, these results demonstrate that PARylation process in Drosophila is tightly regulated in the context of strands-breaks repair and that PARP is essential during the maintenance of DNA integrity but dispensable in the DNA repair process.     

Activation and signaling of the p38 MAP kinase pathway

The family members of the mitogen-activated protein (MAP) kinases mediate a wide variety of cellular behaviors in response to extracellular stimuli. One of the four main sub-groups, the p38 group of MAP kinases, serve as a nexus for signal transduction and play a vital role in numerous biological processes. In this review, we highlight the known characteristics and components of the p38 pathway along with the mechanism and consequences of p38 activation. We focus on the role of p38 as a signal transduction mediator and examine the evidence linking p38 to inflammation, cell cycle, cell death, development, cell differentiation, senescence and tumorigenesis in specific cell types. Upstream and downstream components of p38 are described and questions remaining to be answered are posed. Finally, we propose several directions for future research on p38.     

p38 MAPK signaling pathway is involved in butyrate-induced vitamin D receptor expression

Previously, we have demonstrated that the butyrate-induced differentiation in the human colon cancer cell line Caco-2 occurs via upregulation of the vitamin D receptor (VDR). However, the downstream pathways involved are unknown. The mitogen-activated protein kinases (MAPKs) have been shown to play an important role in regulation of cell differentiation, and may therefore be a potential target of butyrate action. To assess their role in butyrate-mediated cell differentiation and VDR expression, we used the specific p38-MAPK inhibitor SB203580 and the ERK1/2 MAPK-inhibitor PD98059. The p38-MAPK inhibitor abolished the butyrate effect on VDR expression and cell differentiation, while the ERK1/2 inhibitor did not influence the butyrate-mediated induction of cell differentiation and VDR expression. The essential role of the p38 pathway in up-regulation of VDR expression was further confirmed by using the p38 stimulator arsenite. These results imply an important role of the p38-MAPK in regulation of cellular differentiation through upregulation of VDR expression by butyrate.     

50 Years of poly(ADP-ribosyl)ation

The seminal paper published in 1963 by Chambon, Weil and Mandel reporting a new NAD-dependent protein modification now known as poly(ADP-ribosyl)ation (PARylation) marked the launch of a new era in both protein research and cell biology. In the coming decades, the identity, biochemical characteristics and regulation of enzymes responsible for the synthesis and degradation of protein-bound poly(ADP-ribose) have been discovered and the surprisingly multifarious biological roles of PARylation have not ceased to amaze cell and molecular biologists ever since. The review series on PARylation following this preface is comprised of ten papers written by great experts of the field and aims to provide practicing physicians and basic scientists with the state-of-the-art on the “writers, readers and erasers” of poly(ADP-ribose), some recent paradigm shifts of the field and its translational potential.     

Misregulation of poly(ADP-ribose) polymerase-1 activity and cell type-specific loss of poly(ADP-ribose) synthesis in the cerebellum of aged rats

Protein modification by ADP-ribose polymers is a common regulatory mechanism in eukaryotic cells and is involved in several aspects of brain physiology and physiopathology, including neurotransmission, memory formation, neurotoxicity, ageing and age-associated diseases. Here we show age-related misregulation of poly(ADP-ribose) synthesis in rat cerebellum as revealed by: (i) reduced poly(ADP-ribose) polymerase-1 (PARP-1) activation in response to enzymatic DNA cleavage, (ii) altered protein poly(ADP-ribosyl)ation profiles in isolated nuclei, and (iii) cell type-specific loss of poly(ADP-ribosyl)ation capacity in granule cell layer and Purkinje cells in vivo. In particular, although PARP-1 could be detected in virtually all granule cells, only a fraction of them appeared to be actively engaged in poly(ADP-ribose) synthesis and this fraction was reduced in old rat cerebellum. NAD(+), quantified in tissue homogenates, was essentially the same in the cerebellum of young and old rats suggesting that in vivo factors other than PARP-1 content and/or NAD(+) levels may be responsible for the age-associated lowering of poly(ADP-ribose) synthesis. Moreover, PARP-1 expression was substantially down-regulated in Purkinje cells of senescent rats.     

The role of poly(ADP-ribose) polymerases in manganese exposed Caenorhabditis elegans

Background and aimWhen exceeding the homeostatic range, manganese (Mn) might cause neurotoxicity, characteristic of the pathophysiology of several neurological diseases. Although the underlying mechanism of its neurotoxicity remains unclear, Mn-induced oxidative stress contributes to disease etiology. DNA damage caused by oxidative stress may further trigger dysregulation of DNA-damage-induced poly(ADP-ribosyl)ation (PARylation), which is of central importance especially for neuronal homeostasis. Accordingly, this study was designed to assess in the genetically traceable in vivo model Caenorhabditis elegans the role of PARylation as well as the consequences of loss of pme-1 or pme-2 (orthologues of PARP1 and PARP2) in Mn-induced toxicity.MethodsA specific and sensitive isotope-dilution liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed to quantify PARylation in worms. Next to monitoring the PAR level, pme-1 and pme-2 gene expression as well as Mn-induced oxidative stress was studied in wildtype worms and the pme deletion mutants.Results and conclusionWhile Mn failed to induce PARylation in wildtype worms, toxic doses of Mn led to PAR-induction in pme-1-deficient worms, due to an increased gene expression of pme-2 in the pme-1 deletion mutants. However, this effect could not be observed at sub-toxic Mn doses as well as upon longer incubation times. Regarding Mn-induced oxidative stress, the deletion mutants did not show hypersensitivity. Taken together, this study characterizes worms to model PAR inhibition and addresses the consequences for Mn-induced oxidative stress in genetically manipulated worms.     

Human poly(ADP-ribose) glycohydrolase is expressed in alternative splice variants yielding isoforms that localize to different cell compartments

Poly(ADP-ribose) glycohydrolase (PARG) is the only protein known to catalyze hydrolysis of ADP-ribose (ADPR) polymers to free ADP-ribose. While numerous genes encode different poly(ADP-ribose) polymerases (PARPs) that all synthesize ADP-ribose polymer, only a single gene coding for PARG has been detected in mammalian cells. Here, we describe two splice variants of human PARG mRNA, which lead to expression of PARG isoforms of 102 kDa (hPARG102) and 99 kDa (hPARG99) in addition to the full-length PARG protein (hPARG111). These splice variants differ from hPARG111 by the lack of exon 1 (hPARG102) or exons 1 and 2 (hPARG99). They are generated by the utilization of ambiguous splice donor sites in the PARG gene 5' untranslated region. The hPARG111 isoform localizes to the nucleus, whereas hPARG102 and hPARG99 are cytoplasmic proteins. The nuclear targeting of hPARG111 is due to a nuclear localization signal (NLS) in exon 1 that was mapped to the amino acids (aa) (10)CTKRPRW(16). Immunocytochemistry, immunoblotting, and PARG enzyme activity measurements show that the cytoplasmic isoforms of PARG account for most of the PARG activity in cells in the absence and presence of genotoxic stress. The predominantly cytoplasmic location of cellular PARG is intriguing as most known cellular PARPs have a nuclear localization.     

Vibration loading promotes osteogenic differentiation of bone marrow-derived mesenchymal stem cells via p38 MAPK signaling pathway

Low magnitude high frequency vibration (LMHFV) exhibits effectively anabolic effects on the bone tissue, and can promote osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. The role of p38 MAPK signaling in LMHFV-induced osteogenesis remains unclear. In this current study, LMHFV loading was applied to BMSCs in vitro, and cell proliferation, alkaline phosphatase (ALP), matrix mineralization, as well as osteogenic genes expression were assayed. The mechanism of mechanical signal transduction was analysed using PCR array, qRT-PCR and Western blot. LMHFV increased cell proliferation in the growth medium, while inhibited proliferation in the osteogenic medium. ALP activity, matrix mineralization and osteogenic genes expression of Runx2, Col-I, ALP, OPN and OC were increased by LMHFV. p38 and MKK6 genes expression, and p38 phosphorylation were promoted in LMHFV-induced osteogenesis. Inhibition of p38 MAPK with SB203580 and targeted p38 siRNA blunted the increased ALP activity and osteogenic genes expression by LMHFV. These findings suggest that LMHFV promotes osteogenic differentiation of BMSCs, and p38 MAPK signaling shows an important function in LMHFV-induced osteogenesis.     

Apoptotic signaling in dopamine-induced cell death: the role of oxidative stress, p38 mitogen-activated protein kinase, cytochrome c and caspases

Oxidative stress generated by dopamine (DA) oxidation could be one of the factors underlying the selective vulnerability of nigral dopaminergic neurons in Parkinson's diseases. Here we show that DA induces apoptosis in SH-SY5Y neuroblastoma cells demonstrated by activation of caspase-9 and caspase-3, cleavage of poly(ADP-ribose) polymerase as well as nuclear condensation. We also show that p38 mitogen-activated protein kinase is activated within 10 min of DA treatment, which precedes the onset of apoptosis because the potent p38 kinase inhibitor SB203580 protects against DA-induced cell death as well as against caspase-9 and caspase-3 activation. In addition, the antioxidant N-acetyl-L-cysteine (NAC) effectively blocks DA-induced p38 kinase activation, caspase-9 and caspase-3 cleavage and subsequent apoptosis, indicating that DA triggers apoptosis via a signaling pathway that is initiated by the generation of reactive oxygen species (ROS). Dopamine exerts its toxicity principally intracellularly as the DA uptake inhibitor, nomifensine significantly reduces DA-induced cell death as well as activation of p38 kinase and caspase-3. Furthermore, DA induces mitochondrial cytochrome c release, which is dependent on p38 kinase activation and precedes the cleavage of caspases. These observations indicate that DA induces apoptosis primarily by generating ROS, p38 kinase activation, cytochrome c release followed by caspase-9 and caspase-3 activation.     

Poly(ADP-ribose) signaling in cell death

Poly(ADP-ribosyl)ation (PARylation) is a reversible protein modification carried out by the concerted actions of poly(ADP-ribose) polymerase (PARP) enzymes and poly(ADP-ribose) (PAR) decomposing enzymes such as PAR glycohydrolase (PARG) and ADP-ribosyl hydrolase 3 (ARH3). Reversible PARylation is a pleiotropic regulator of various cellular functions but uncontrolled PARP activation may also lead to cell death. The cellular demise pathway mediated by PARylation in oxidatively stressed cells has been described almost thirty years ago. However, the underlying molecular mechanisms have only begun to emerge relatively recently. PARylation has been implicated in necroptosis, autophagic cell death but its role in extrinsic and intrinsic apoptosis appears to be less predominant and depends largely on the cellular model used. Currently, three major pathways have been made responsible for PARP-mediated necroptotic cell death: (1) compromised cellular energetics mainly due to depletion of NAD, the substrate of PARPs; (2) PAR mediated translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus (parthanatos) and (3) a mostly elusive crosstalk between PARylation and cell death/survival kinases and phosphatases. Here we review how these PARP-mediated necroptotic pathways are intertwined, how PARylation may contribute to extrinsic and intrinsic apoptosis and discuss recent developments on the role of PARylation in autophagy and autophagic cell death.     

The role of p38 MAP kinase and c-Jun N-terminal protein kinase signaling in the differentiation and apoptosis of immortalized neural stem cells

The two distinct members of the mitogen-activated protein (MAP) kinase family c-Jun N-terminal protein kinase (JNK) and p38 MAP kinase, play an important role in central nervous system (CNS) development and differentiation. However, their role and functions are not completely understood in CNS. To facilitate in vitro study, we have established an immortal stem cell line using SV40 from fetal rat embryonic day 17. In these cells, MAP kinase inhibitors (SP600125, SB202190, and PD98059) were treated for 1, 24, 48, and 72 h to examine the roles of protein kinases. Early inhibition of JNK did not alter phenotypic or morphological changes of immortalized cells, however overexpression of Bax and decrease of phosphorylated AKT was observed. The prolonged inhibition of JNK induced polyploidization of immortalized cells, and resulted in differentiation and inhibition of cell proliferation. Moreover, JNK and p38 MAP kinase but not ERK1/2 was activated, and p21, p53, and Bax were overexpressed by prolonged inhibition of JNK.

These results indicate that JNK and p38 MAP kinase could play dual roles on cell survival and apoptosis. Furthermore, this established cell line could facilitate study of the role of JNK and p38 MAP kinase on CNS development or differentiation/apoptosis.     

Honokiol causes the p21WAF1-mediated G(1)-phase arrest of the cell cycle through inducing p38 mitogen activated protein kinase in vascular smooth muscle cells

Honokiol, an active component in extracts of Magnolia officinalis, has been proposed to play a role in anti-inflammatory, antioxidant activity, anti-angiogenic and anti-tumor activity. Although honokiol has a variety of pharmacological effects on certain cell types, its effects on vascular smooth muscle cells (VSMC) are unclear. This issue was investigated in the present study, honokiol was found to inhibit cell viability and DNA synthesis in cultured VSMC. These inhibitory effects were associated with G1 cell cycle arrest. Treatment with honokiol blocks the cell cycle in the G1 phase, down-regulates the expression of cyclins and CDKs and up-regulates the expression of p21WAF1, a CDK inhibitor. While honokiol did not up-regulate p27, it caused an increase in the promoter activity of the p21WAF1 gene. Immunoblot and deletion analysis of the p21WAF1 promoter showed that honokiol induced the expression of p21WAF1 and that this expression was independent of the p53 pathway. Furthermore, the honokiol-mediated signaling pathway involved in VSMC growth inhibition was examined. Among the relevant pathways, honokiol induced a marked activation of p38 MAP kinase and JNK. The expression of dominant negative p38 MAP kinase and SB203580, a p38 MAP kinase specific inhibitor, blocked the expression of honokiol-dependent p38 MAP kinase and p21WAF1. Consistently, blockade of p38 MAPK kinase function reversed honokiol-induced VSMC proliferation and cell cycle proteins. These data demonstrate that the p38 MAP kinase pathway participates in p21WAF1 induction, subsequently leading to a decrease in the levels of cyclin D1/CDK4 and cyclin E/CDK2 complexes and honokiol-dependent VSMC growth inhibition. In conclusion, these findings concerning the molecular mechanisms of honokiol in VSMC provides a theoretical basis for clinical approaches to the use therapeutic agents in treating atherosclerosis.     

Blockade of PARP activity attenuates poly(ADP-ribosyl)ation but offers only partial neuroprotection against NMDA-induced cell death in the rat retina

Recent reports have linked neuronal cell death by necrosis to poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation. It is believed that under stress, the activity of this enzyme is up-regulated, resulting in extensive poly(ADP-ribosyl)ation of nuclear proteins, using NAD(+) as its substrate, which, in turn, leads to the depletion of NAD(+). In efforts to restore the level of NAD(+), depletion of ATP occurs, resulting in the shutdown of ATP-dependent ionic pumps. This results in cell swelling and eventual loss of membrane selectivity, hallmarks of necrosis. Reports from in vitro and in vivo studies in the brain have shown that NMDA receptor activation stimulates PARP activity and that blockade of the enzyme provides substantial neuroprotection. The present study was undertaken to determine whether PARP activity is regulated by NMDA in the rat retina, and whether blockade of PARP activity provides protection against toxic effects of NMDA. Rat retinas exposed to intravitreal injections containing NMDA, with or without the PARP inhibitor N-(6-oxo-5, 6-dihydrophenanthridin-2-yl)-(N,-dimethylamino) acetamide hydrochloride (PJ-34), were assessed for changes in PARP-1 activity as evidenced by poly(ADP-ribosyl)ation (PAR), loss of membrane integrity, morphological indicators of apoptosis and necrosis, and ganglion cell loss. Results showed that: NMDA increased PAR formation in a concentration-dependent manner and caused a decline in retinal ATP levels; PJ-34 blockade attenuated the NMDA-induced formation of PAR and decline in ATP; NMDA induced the loss of membrane selectivity to ethidium bromide (EtBr) in inner retinal neurons, but loss of membrane selectivity was not prevented by blocking PARP activity; cells stained with EtBr, or reacted for TUNEL-labeling, displayed features characteristic of both apoptosis and necrosis. In the presence of PJ-34, greater numbers of cells exhibited apoptotic features; PJ-34 provided partial neuroprotection against NMDA-induced ganglion cell loss. These findings suggest that although blockade of PARP activity fully attenuates NMDA-induced PAR formation and loss of retinal ATP content, and improves the survival of select populations of ganglion cells, this approach does not provide full neuroprotection. In contrast, blockade of PARP activity promotes apoptotic-like cell death in the majority of cells undergoing cell death. Furthermore, these studies show that the loss of membrane selectivity is not dependent upon PAR formation or the resulting decline of ATP, and suggests that an alternative pathway, other than PARP activation, exists to mediate this event.     

Design and synthesis of potent inhibitors of the mono(ADP-ribosyl)transferase,PARP14

A series of (Z)-4-(3-carbamoylphenylamino)-4-oxobut-2-enyl amides were synthesized and tested for their ability to inhibit the mono-(ADP-ribosyl)transferase, PARP14 (a.k.a. BAL-2; ARTD-8). Two synthetic routes were established for this series and several compounds were identified as sub-micromolar inhibitors of PARP14, the most potent of which was compound 4t, IC₅₀ = 160 nM. Furthermore, profiling other members of this series identified compounds with >20-fold selectivity over PARP5a/TNKS1, and modest selectivity over PARP10, a closely related mono-(ADP-ribosyl)transferase.     

MKK6-p38 MAPK signaling pathway enhances survival but not bone-resorbing activity of osteoclasts

Phosphorylated p38 mitogen-activating kinase (MAPK) is observed in osteoclasts under in vivo inflammatory situations. However, the role of p38 MAPK in osteoclast function has not been elucidated, because all external stimuli tested hitherto failed to induce the phosphorylation of p38 MAPK in osteoclasts in culture. In this study, a constitutively active form of MKK6 (MKK6CA) was expressed in osteoclasts using adenoviral gene transfer in vitro. MKK6CA expressed in osteoclasts phosphorylated p38 MAPK and enhanced the survival of osteoclasts. Dentine-resorbing activity of osteoclasts was not enhanced by the MKK6CA expression. These results suggest that p38 MAPK signaling plays a critical role in the survival of osteoclasts in inflammatory diseases.