Correction: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3'-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis.

[This corrects the article DOI: 10.1371/journal.pgen.1004749.].

Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways.

Mammalian polynucleotide kinase 3'-phosphatase (PNKP), a DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP's enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP's role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.

8-Oxoguanine targeted by 8-oxoguanine DNA glycosylase 1 (OGG1) is central to fibrogenic gene activation upon lung injury.

Reactive oxygen species (ROS) are implicated in epithelial cell-state transition and deposition of extracellular matrix upon airway injury. Of the many cellular targets of ROS, oxidative DNA modification is a major driving signal. However, the role of oxidative DNA damage in modulation profibrotic processes has not been fully delineated. Herein, we report that oxidative DNA base lesions, 8-oxoG, complexed with 8-oxoguanine DNA glycosylase 1 (OGG1) functions as a pioneer factor, contributing to transcriptional reprogramming within airway epithelial cells. We show that TGFß1-induced ROS increased 8-oxoG levels in open chromatin, dynamically reconfigure the chromatin state. OGG1 complexed with 8-oxoG recruits transcription factors, including phosphorylated SMAD3, to pro-fibrotic gene promoters thereby facilitating gene activation. Moreover, 8-oxoG levels are elevated in lungs of mice subjected to TGFß1-induced injury. Pharmacologic targeting of OGG1 with the selective small molecule inhibitor of 8-oxoG binding, TH5487, abrogates fibrotic gene expression and remodeling in this model. Collectively, our study implicates that 8-oxoG substrate-specific binding by OGG1 is a central modulator of transcriptional regulation in response to tissue repair.

Functional analysis of a conserved site mutation in the DNA end processing enzyme PNKP leading to ataxia with oculomotor apraxia type 4 in humans.

Polynucleotide kinase 3'-phosphatase (PNKP), an essential DNA end-processing enzyme in mammals with 3'-phosphatase and 5'-kinase activities, plays a pivotal role in multiple DNA repair pathways. Its functional deficiency has been etiologically linked to various neurological disorders. Recent reports have shown that mutation at a conserved glutamine (Gln) in PNKP leads to late-onset ataxia with oculomotor apraxia type 4 (AOA4) in humans and embryonic lethality in pigs. However, the molecular mechanism underlying such phenotypes remains elusive. Here, we report that the enzymatic activities of the mutant versus WT PNKP are comparable; however, cells expressing mutant PNKP and peripheral blood mononuclear cells (PBMCs) of AOA4 patients showed a significant amount of DNA double-strand break accumulation and consequent activation of the DNA damage response. Further investigation revealed that the nuclear localization of mutant PNKP is severely abrogated, and the mutant proteins remain primarily in the cytoplasm. Western blot analysis of AOA4 patient-derived PBMCs also revealed the presence of mutated PNKP predominantly in the cytoplasm. To understand the molecular determinants, we identified that mutation at a conserved Gln residue impedes the interaction of PNKP with importin alpha but not with importin beta, two highly conserved proteins that mediate the import of proteins from the cytoplasm into the nucleus. Collectively, our data suggest that the absence of PNKP in the nucleus leads to constant activation of the DNA damage response due to persistent accumulation of double-strand breaks in the mutant cells, triggering death of vulnerable brain cells-a potential cause of neurodegeneration in AOA4 patients.

Nei-like DNA glycosylase 2 selectively antagonizes interferon-ß expression upon respiratory syncytial virus infection.

As part of the antiviral response, cells activate the expressions of type I interferons (IFNs) and proinflammatory mediators to control viral spreading. Viral infections can impact DNA integrity; however, how DNA damage repair coordinates antiviral response remains elusive. Here we report Nei-like DNA glycosylase 2 (NEIL2), a transcription-coupled DNA repair protein, actively recognizes the oxidative DNA substrates induced by respiratory syncytial virus (RSV) infection to set the threshold of IFN-ß expression. Our results show that NEIL2 antagonizes nuclear factor κB (NF-κB) acting on the IFN-ß promoter early after infection, thus limiting gene expression amplified by type I IFNs. Mice lacking Neil2 are far more susceptible to RSV-induced illness with an exuberant expression of proinflammatory genes and tissue damage, and the administration of NEIL2 protein into the airway corrected these defects. These results suggest a safeguarding function of NEIL2 in controlling IFN-ß levels against RSV infection. Due to the short- and long-term side effects of type I IFNs applied in antiviral therapy, NEIL2 may provide an alternative not only for ensuring genome fidelity but also for controlling immune responses.

8-Oxoguanine DNA glycosylase 1 selectively modulates ROS-responsive NF-κB targets through recruitment of MSK1 and phosphorylation of RelA/p65 at Ser276.

Nuclear factor kappa B (NF-κB) activity is regulated by various posttranslational modifications, of which Ser276 phosphorylation of RelA/p65 is particularly impacted by reactive oxygen species (ROS). This modification is responsible for selective upregulation of a subset of NF-κB targets; however, the precise mechanism remains elusive. ROS have the ability to modify cellular molecules including DNA. One of the most common oxidation products is 8-oxo-7,8-dihydroguanine (8-oxoGua), which is repaired by the 8-oxoguanine DNA glycosylase1 (OGG1)-initiated base excision repair pathway. Recently, a new function of OGG1 has been uncovered. OGG1 binds to 8-oxoGua, facilitating the occupancy of NF-κB at promoters and enhancing transcription of pro-inflammatory cytokines and chemokines. In the present study, we demonstrated that an interaction between DNA-bound OGG1 and mitogen-and stress-activated kinase 1 is crucial for RelA/p65 Ser276 phosphorylation. ROS scavenging or OGG1 depletion/inhibition hindered the interaction between mitogen-and stress-activated kinase 1 and RelA/p65, thereby decreasing the level of phospho-Ser276 and leading to significantly lowered expression of ROS-responsive cytokine/chemokine genes, but not that of Nfkbis. Blockade of OGG1 binding to DNA also prevented promoter recruitment of RelA/p65, Pol II, and p-RNAP II in a gene-specific manner. Collectively, the data presented offer new insights into how ROS signaling dictates NF-κB phosphorylation codes and how the promoter-situated substrate-bound OGG1 is exploited by aerobic mammalian cells for timely transcriptional activation of ROS-responsive genes.

Cell-Penetrating Peptide TAT-HuR-HNS3 Suppresses Proinflammatory Gene Expression via Competitively Blocking Interaction of HuR with Its Partners.

Proinflammatory cytokines/chemokines are commonly regulated by RNA-binding proteins at posttranscriptional levels. Human Ag R (HuR)/embryonic lethal abnormal vision-like 1 (ELAVL1) is one of the well-characterized RNA-binding proteins that increases the stability of short-lived mRNAs, which encode proinflammatory mediators. HuR employs its nucleocytoplasmic shuttling sequence (HNS) domain, interacting with poly(ADP-ribose) polymerase 1 (PARP1), which accounts for the enhanced poly-ADP-ribosylation and cytoplasmic shuttling of HuR. Also by using its HNS domain, HuR undergoes dimerization/oligomerization, underlying the increased binding of HuR with proinflammatory cytokine/chemokine mRNAs and the disassociation of the miRNA-induced silencing complex from the targets. Therefore, competitively blocking the interactions of HuR with its partners may suppress proinflammatory mediator production. In this study, peptides derived from the sequence of the HuR-HNS domain were synthesized, and their effects on interfering HuR interacting with PARP1 and HuR itself were analyzed. Moreover, cell-penetrating TAT-HuR-HNS3 was delivered into human and mouse cells or administered into mouse lungs with or without exposure of TNF-α or LPS. mRNA levels of proinflammatory mediators as well as neutrophil infiltration were evaluated. We showed that TAT-HuR-HNS3 interrupts HuR-PARP1 interaction and therefore results in a lowered poly-ADP-ribosylation level and decreased cytoplasmic distribution of HuR. TAT-HuR-HNS3 also blocks HuR dimerization and promotes Argonaute 2-based miRNA-induced silencing complex binding to the targets. Moreover, TAT-HuR-HNS3 lowers mRNA stability of proinflammatory mediators in TNF-α-treated epithelial cells and macrophages, and it decreases TNF-α-induced inflammatory responses in lungs of experimental animals. Thus, TAT-HuR-HNS3 is a promising lead peptide for the development of inhibitors to treat inflammation-related diseases.

No strong association among epigenetic modifications by DNA methylation, telomere length, and physical fitness in biological aging.

Cellular senescence is greatly accelerated by telomere shortening, and the steps forward in human aging are strongly influenced by environmental and lifestyle factors, whether DNA methylation (DNAm) is affected by exercise training, remains unclear. In the present study, we investigated the relationships between physiological functions, maximal oxygen uptake (VO2max), vertical jump, working memory, telomere length (TL) assessed by RT-PCR, DNA methylation-based estimation of TL (DNAmTL), and DNA methylation-based biomarkers of aging of master rowers (N = 146) and sedentary subjects (N = 95), aged between 37 and 85 years. It was found that the TL inversely correlated with chronological age. We could not detect an association between telomere length and VO2max, vertical jump, and working memory by RT-PCR method, while these physiological test results showed a correlation with DNAmTL. DNAmGrimAge and DNAmPhenoAge acceleration were inversely associated with telomere length assessed by both methods. It appears that there are no strong beneficial effects of exercise or physiological fitness on telomere shortening, however, the degree of DNA methylation is associated with telomere length.

Intrapulmonary administration of purified NEIL2 abrogates NF-κB-mediated inflammation.

Aberrant or constitutive activation of nuclear factor kappa B (NF-κB) contributes to various human inflammatory diseases and malignancies via the upregulation of genes involved in cell proliferation, survival, angiogenesis, inflammation, and metastasis. Thus, inhibition of NF-κB signaling has potential for therapeutic applications in cancer and inflammatory diseases. We reported previously that Nei-like DNA glycosylase 2 (NEIL2), a mammalian DNA glycosylase, is involved in the preferential repair of oxidized DNA bases from the transcriptionally active sequences via the transcription-coupled base excision repair pathway. We have further shown that Neil2-null mice are highly sensitive to tumor necrosis factor α (TNFα)- and lipopolysaccharide-induced inflammation. Both TNFα and lipopolysaccharide are potent activators of NF-κB. However, the underlying mechanism of NEIL2's role in the NF-κB-mediated inflammation remains elusive. Here, we have documented a noncanonical function of NEIL2 and demonstrated that the expression of genes, such as Cxcl1, Cxcl2, Cxcl10, Il6, and Tnfα, involved in inflammation and immune cell migration was significantly higher in both mock- and TNFα-treated Neil2-null mice compared with that in the WT mice. NEIL2 blocks NF-κB's binding to target gene promoters by directly interacting with the Rel homology region of RelA and represses proinflammatory gene expression as determined by co-immunoprecipitation, chromatin immunoprecipitation, and electrophoretic mobility-shift assays. Remarkably, intrapulmonary administration of purified NEIL2 via a noninvasive nasal route significantly abrogated binding of NF-κB to cognate DNA, leading to decreased expression of proinflammatory genes and neutrophil recruitment in Neil2-null as well as WT mouse lungs. Our findings thus highlight the potential of NEIL2 as a biologic for inflammation-associated human diseases.

Poly(ADP-ribosyl)ation enhances HuR oligomerization and contributes to pro-inflammatory gene mRNA stabilization.

Poly(ADP-ribosyl)ation (PARylation) is an important post-translational modification mainly catalyzed by poly-ADP-ribose polymerase 1 (PARP1). In addition to having important roles in DNA damage detection and repair, it functions in gene expression regulation, especially at the posttranscriptional level. Embryonic lethal abnormal vision-like 1/human antigen R (ELAVL/HuR), a canonical 3' untranslated region AU-rich element-binding protein, is a crucial mRNA-stabilizing protein that protects target mRNAs from RNA-destabilizing protein- or microRNA-induced silencing complex (miRISC)-mediated degradation. Additionally, in some cases, HuR itself either promotes or suppresses translation. Here, we demonstrated that in response to inflammatory stimuli, the PARylation of HuR, mostly at the conserved D226 site, by PARP1 increased the formation of the HuR oligomer/multimer, and HuR oligomerization promoted the disassociation of miRISC and stabilized the pro-inflammatory gene mRNAs. The prevention of PARP1 activation or HuR oligomerization attenuated lipopolysaccharide-induced inflammatory gene expression and the airway recruitment of neutrophils in mouse lungs. The present study verified a novel mechanism of PARP1 and HuR PARylation in the RNA stability regulation, increasing our understanding of how PARP1 regulates gene expression.

Motor neuron disease-associated loss of nuclear TDP-43 is linked to DNA double-strand break repair defects.

Genome damage and their defective repair have been etiologically linked to degenerating neurons in many subtypes of amyotrophic lateral sclerosis (ALS) patients; however, the specific mechanisms remain enigmatic. The majority of sporadic ALS patients feature abnormalities in the transactivation response DNA-binding protein of 43 kDa (TDP-43), whose nucleo-cytoplasmic mislocalization is characteristically observed in spinal motor neurons. While emerging evidence suggests involvement of other RNA/DNA binding proteins, like FUS in DNA damage response (DDR), the role of TDP-43 in DDR has not been investigated. Here, we report that TDP-43 is a critical component of the nonhomologous end joining (NHEJ)-mediated DNA double-strand break (DSB) repair pathway. TDP-43 is rapidly recruited at DSB sites to stably interact with DDR and NHEJ factors, specifically acting as a scaffold for the recruitment of break-sealing XRCC4-DNA ligase 4 complex at DSB sites in induced pluripotent stem cell-derived motor neurons. shRNA or CRISPR/Cas9-mediated conditional depletion of TDP-43 markedly increases accumulation of genomic DSBs by impairing NHEJ repair, and thereby, sensitizing neurons to DSB stress. Finally, TDP-43 pathology strongly correlates with DSB repair defects, and damage accumulation in the neuronal genomes of sporadic ALS patients and in Caenorhabditis elegans mutant with TDP-1 loss-of-function. Our findings thus link TDP-43 pathology to impaired DSB repair and persistent DDR signaling in motor neuron disease, and suggest that DSB repair-targeted therapies may ameliorate TDP-43 toxicity-induced genome instability in motor neuron disease.

Enzymatically inactive OGG1 binds to DNA and steers base excision repair toward gene transcription.

8-Oxoguanine DNA glycosylase1 (OGG1)-initiated base excision repair (BER) is the primary pathway to remove the pre-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Recent studies documented 8-oxoG serves as an epigenetic-like mark and OGG1 modulates gene expression in oxidatively stressed cells. For this new role of OGG1, two distinct mechanisms have been proposed: one is coupled to base excision, while the other only requires substrate binding of OGG1--both resulting in conformational adjustment in the adjacent DNA sequences providing access for transcription factors to their cis-elements. The present study aimed to examine if BER activity of OGG1 is required for pro-inflammatory gene expression. To this end, Ogg1/OGG1 knockout/depleted cells were transfected with constructs expressing wild-type (wt) and repair-deficient mutants of OGG1. OGG1's promoter enrichment, oxidative state, and gene expression were examined. Results showed that TNFα exposure increased levels of oxidatively modified cysteine(s) of wt OGG1 without impairing its association with promoter and facilitated gene expression. The excision deficient K249Q mutant was even a more potent activator of gene expression; whereas, mutant OGG1 with impaired substrate recognition/binding was not. These data suggested the interaction of OGG1 with its substrate at regulatory regions followed by conformational adjustment in the adjacent DNA is the primary mode to modulate inflammatory gene expression.

c-Abl-Mediated Tyrosine Phosphorylation of PARP1 Is Crucial for Expression of Proinflammatory Genes.

Poly(ADP-ribosyl)ation is a rapid and transient posttranslational protein modification mostly catalyzed by poly(ADP-ribose) polymerase-1 (PARP1). Fundamental roles of activated PARP1 in DNA damage repair and cellular response pathways are well established; however, the precise mechanisms by which PARP1 is activated independent of DNA damage, and thereby playing a role in expression of inflammatory genes, remain poorly understood. In this study, we show that, in response to LPS or TNF-α exposure, the nonreceptor tyrosine kinase c-Abl undergoes nuclear translocation and interacts with and phosphorylates PARP1 at the conserved Y829 site. Tyrosine-phosphorylated PARP1 is required for protein poly(ADP-ribosyl)ation of RelA/p65 and NF-κB-dependent expression of proinflammatory genes in murine RAW 264.7 macrophages, human monocytic THP1 cells, or mouse lungs. Furthermore, LPS-induced airway lung inflammation was reduced by inhibition of c-Abl activity. The present study elucidated a novel signaling pathway to activate PARP1 and regulate gene expression, suggesting that blocking the interaction of c-Abl with PARP1 or pharmaceutical inhibition of c-Abl may improve the outcomes of PARP1 activation-mediated inflammatory diseases.

Epigenetic regulation of TIMP1 expression by 8-oxoguanine DNA glycosylase-1 binding to DNA:RNA hybrid.

8-Oxoguanine DNA glycosylase-1 (OGG1)-initiated base excision repair pathway is primarily responsible for 7, 8-dihydro-8-oxoguanine (8-oxoG) removal from DNA. Recent studies, however, have shown that 8-oxoG in gene regulatory elements may serve as an epigenetic mark, and OGG1 has distinct functions in modulating gene expression. Genome-wide mapping of oxidative stress-induced OGG1 enrichment within introns was documented, but its significance has not yet been fully characterized. Here, we explored whether OGG1 recruited to intron 1 of tissue inhibitor of metalloproteinase-1 (TIMP1) gene and modulated its expression. Using chromatin and DNA:RNA hybrid immunoprecipitation assays, we report recruitment of OGG1 to the DNA:RNA hybrid in intron 1, where it increases nascent RNA but lowers mRNA levels in O3-exposed human airway epithelial cells and mouse lungs. Decrease in TIMP1 expression is alleviated by antioxidant administration, small interfering RNA depletion, or inhibition of OGG1 binding to its genomic substrate. In vitro studies revealed direct interaction between OGG1 and 8-oxoG containing DNA:RNA hybrid, without excision of its substrate. Inhibition of OGG1 binding to DNA:RNA hybrid translated into an increase in TIMP1 expression and a decrease in oxidant-induced lung inflammatory responses as well as airway remodeling. Data documented here reveal a novel molecular link between OGG1 at damaged sites and transcription dynamics that may contribute to oxidative stress-induced cellular and tissue responses.-Pan, L., Wang, H., Luo, J., Zeng, J., Pi, J., Liu, H., Liu, C., Ba, X., Qu, X., Xiang, Y., Boldogh, I., Qin, X. Epigenetic regulation of TIMP1 expression by 8-oxoguanine DNA glycosylase-1 binding to DNA:RNA hybrid.

The roles of base excision repair enzyme OGG1 in gene expression.

Modifications of DNA strands and nucleobases-both induced and accidental-are associated with unfavorable consequences including loss or gain in genetic information and mutations. Therefore, DNA repair proteins have essential roles in keeping genome fidelity. Recently, mounting evidence supports that 8-oxoguanine (8-oxoG), one of the most abundant genomic base modifications generated by reactive oxygen and nitrogen species, along with its cognate repair protein 8-oxoguanine DNA glycosylase1 (OGG1), has distinct roles in gene expression through transcription modulation or signal transduction. Binding to 8-oxoG located in gene regulatory regions, OGG1 acts as a transcription modulator, which can control transcription factor homing, induce allosteric transition of G-quadruplex structure, or recruit chromatin remodelers. In addition, post-repair complex formed between OGG1 and its repair product-free 8-oxoG increases the levels of active small GTPases and induces downstream signaling cascades to trigger gene expressions. The present review discusses how cells exploit damaged guanine base(s) and the authentic repair protein to orchestrate a profile of various transcriptomes in redox-regulated biological processes.

Excision release of 5?hydroxycytosine oxidatively induced DNA base lesions from the lung genome by cat dander extract challenge stimulates allergic airway inflammation.

BACKGROUND: Ragweed pollen extract (RWPE) induces TLR4-NFκB-CXCL-dependent recruitment of ROS-generating neutrophils to the airway and OGG1 DNA glycosylase-dependent excision of oxidatively induced 8-OH-Gua DNA base lesions from the airway epithelial cell genome. Administration of free 8-OH-Gua base stimulates RWPE-induced allergic lung inflammation. These studies suggest that stimulation of innate receptors and their adaptor by allergenic extracts initiates excision of a set of DNA base lesions that facilitate innate/allergic lung inflammation. OBJECTIVE: To test the hypothesis that stimulation of a conserved innate receptor/adaptor pathway by allergenic extracts induces excision of a set of pro-inflammatory oxidatively induced DNA base lesions from the lung genome that stimulate allergic airway inflammation. METHODS: Wild-type (WT), Tlr4KO, Tlr2KO, Myd88KO, and TrifKO mice were intranasally challenged once or repeatedly with cat dander extract (CDE), and innate or allergic inflammation and gene expression were quantified. We utilized GC-MS/MS to quantify a set of oxidatively induced DNA base lesions after challenge of naïve mice with CDE. RESULTS: A single CDE challenge stimulated innate neutrophil recruitment that was partially dependent on TLR4 and TLR2, and completely on Myd88, but not TRIF. A single CDE challenge stimulated MyD88-dependent excision of DNA base lesions 5-OH-Cyt, FapyAde, and FapyGua from the lung genome. A single challenge of naïve WT mice with 5-OH-Cyt stimulated neutrophilic lung inflammation. Multiple CDE instillations stimulated MyD88-dependent allergic airway inflammation. Multiple administrations of 5-OH-Cyt with CDE stimulated allergic sensitization and allergic airway inflammation. CONCLUSIONS AND CLINICAL RELEVANCE: We show for the first time that CDE challenge stimulates MyD88-dependent excision of DNA base lesions. Our data suggest that the resultant-free base(s) contribute to CDE-induced innate/allergic lung inflammation. We suggest that blocking the MyD88 pathway in the airways with specific inhibitors may be a novel targeted strategy of inhibiting amplification of innate and adaptive immune inflammation in allergic diseases by oxidatively induced DNA base lesions.

The establishment of methods for free PAR generation and PAR reader detection.

Poly (ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor that catalyzes the poly (ADP-ribose) (PAR) onto a variety of target proteins, such as histones, DSB repair factors and PARP1 itself under consumption of NAD+. Besides, PARP1 can affect a variety of proteins in noncovalent modification manner to carry out specific cellular functions. Here, we established a method to generate non-radiolabeled free PAR by PARG moderately cleaving PAR from autoPARylated PARP1, and utilized dot-blot assay to determine the interaction between free PAR and interested proteins. The methods to generate free PAR and detect the noncovalent interactions between proteins and free PAR are nonradioactive and convenient, which will facilitate the studies to explore the significance of PAR reading in various biological processes.

The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3'-phosphatase in spinocerebellar ataxia type 3 pathogenesis.

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

NF-κB Mediates Mesenchymal Transition, Remodeling, and Pulmonary Fibrosis in Response to Chronic Inflammation by Viral RNA Patterns.

Airway remodeling is resultant of a complex multicellular response associated with a progressive decline of pulmonary function in patients with chronic airway disease. Here, repeated infections with respiratory viruses are linked with airway remodeling through largely unknown mechanisms. Although acute activation of the Toll-like receptor (TLR) 3 pathway by extracellular polyinosinic:polycytidylic acid (poly[I:C]) induces innate signaling through the NF-κB transcription factor in normal human small airway epithelial cells, prolonged (repetitive or tonic) poly(I:C) stimulation produces chronic stress fiber formation, mesenchymal transition, and activation of a fibrotic program. Chronic poly(I:C) stimulation enhanced the expression of core mesenchymal regulators Snail family zinc finger 1, zinc finger E-box binding homeobox, mesenchymal intermediate filaments (vimentin), and extracellular matrix proteins (fibronectin-1), and collagen 1A. This mesenchymal transition was prevented by silencing expression of NF-κB/RelA or administration of a small-molecule inhibitor of the IκB kinase, BMS345541. Acute poly(I:C) exposure in vivo induced profound neutrophilic airway inflammation. When administered repetitively, poly(I:C) resulted in enhanced fibrosis observed by lung micro-computed tomography, second harmonic generation microscopy of optically cleared lung tissue, and by immunohistochemistry. Epithelial flattening, expansion of the epithelial mesenchymal trophic unit, and enhanced Snail family zinc finger 1 and fibronectin 1 expression in airway epithelium were also observed. Repetitive poly(I:C)-induced airway remodeling, fibrosis, and epithelial-mesenchymal transition was inhibited by BMS345541 administration. Based on this novel model of viral inflammation-induced remodeling, we conclude that NF-κB is a major controller of epithelial-mesenchymal transition and pulmonary fibrosis, a finding that has potentially important relevance to airway remodeling produced by repetitive viral infections.

SIRT1 may play a crucial role in overload-induced hypertrophy of skeletal muscle.

KEY POINTS: Silent mating type information regulation 2 homologue 1 (SIRT1) activity and content increased significantly in overload-induced hypertrophy. SIRT1-mediated signalling through Akt, the endothelial nitric oxide synthase mediated pathway, regulates anabolic process in the hypertrophy of skeletal muscle. The regulation of catabolic signalling via forkhead box O 1 and protein ubiquitination is SIRT1 dependent. Overload-induced changes in microRNA levels regulate SIRT1 and insulin-like growth factor 1 signalling. ABSTRACT: Significant skeletal muscle mass guarantees functional wellbeing and is important for high level performance in many sports. Although the molecular mechanism for skeletal muscle hypertrophy has been well studied, it still is not completely understood. In the present study, we used a functional overload model to induce plantaris muscle hypertrophy by surgically removing the soleus and gastrocnemius muscles in rats. Two weeks of muscle ablation resulted in a 40% increase in muscle mass, which was associated with a significant increase in silent mating type information regulation 2 homologue 1 (SIRT1) content and activity (P < 0.001). SIRT1-regulated Akt, endothelial nitric oxide synthase and GLUT4 levels were also induced in hypertrophied muscles, and SIRT1 levels correlated with muscle mass, paired box protein 7 (Pax7), proliferating cell nuclear antigen (PCNA) and nicotinamide phosphoribosyltransferase (Nampt) levels. Alternatively, decreased forkhead box O 1 (FOXO1) and increased K48 polyubiquitination also suggest that SIRT1 could be involved in the catabolic process of hypertrophy. Furthermore, increased levels of K63 and muscle RING finger 2 (MuRF2) protein could also be important enhancers of muscle mass. We report here that the levels of miR1 and miR133a decrease in hypertrophy and negatively correlate with muscle mass, SIRT1 and Nampt levels. Our results reveal a strong correlation between SIRT1 levels and activity, SIRT1-regulated pathways and overload-induced hypertrophy. These findings, along with the well-known regulatory roles that SIRT1 plays in modulating both anabolic and catabolic pathways, allow us to propose the hypothesis that SIRT1 may actually play a crucial causal role in overload-induced hypertrophy of skeletal muscle. This hypothesis will now require rigorous direct and functional testing.