Light-strand bias and enriched zones of embedded ribonucleotides are associated with DNA replication and transcription in the human-mitochondrial genome.

Abundant ribonucleoside-triphosphate (rNTP) incorporation into DNA by DNA polymerases in the form of ribonucleoside monophosphates (rNMPs) is a widespread phenomenon in nature, resulting in DNA-structural change and genome instability. The rNMP distribution, characteristics, hotspots and association with DNA metabolic processes in human mitochondrial DNA (hmtDNA) remain mostly unknown. Here, we utilize the ribose-seq technique to capture embedded rNMPs in hmtDNA of six different cell types. In most cell types, the rNMPs are preferentially embedded on the light strand of hmtDNA with a strong bias towards rCMPs; while in the liver-tissue cells, the rNMPs are predominately found on the heavy strand. We uncover common rNMP hotspots and conserved rNMP-enriched zones across the entire hmtDNA, including in the control region, which links the rNMP presence to the frequent hmtDNA replication-failure events. We show a strong correlation between coding-sequence size and rNMP-embedment frequency per nucleotide on the non-template, light strand in all cell types, supporting the presence of transient RNA-DNA hybrids preceding light-strand replication. Moreover, we detect rNMP-embedment patterns that are only partly conserved across the different cell types and are distinct from those found in yeast mtDNA. The study opens new research directions to understand the biology of hmtDNA and genomic rNMPs.

Is Drp1 a link between mitochondrial dysfunction and inflammation in Alzheimer's disease?

Recent advances highlight that inflammation is critical to Alzheimer Disease (AD) pathogenesis. Indeed, several diseases characterized by inflammation are considered risk factors for AD, such as type 2 diabetes, obesity, hypertension, and traumatic brain injury. Moreover, allelic variations in genes involved in the inflammatory cascade are risk factors for AD. AD is also characterized by mitochondrial dysfunction, which affects the energy homeostasis of the brain. The role of mitochondrial dysfunction has been characterized mostly in neuronal cells. However, recent data are demonstrating that mitochondrial dysfunction occurs also in inflammatory cells, promoting inflammation and the secretion of pro-inflammatory cytokines, which in turn induce neurodegeneration. In this review, we summarize the recent finding supporting the hypothesis of the inflammatory-amyloid cascade in AD. Moreover, we describe the recent data that demonstrate the link between altered mitochondrial dysfunction and the inflammatory cascade. We focus in summarizing the role of Drp1, which is involved in mitochondrial fission, showing that altered Drp1 activation affects the mitochondrial homeostasis and leads to the activation of the NLRP3 inflammasome, promoting the inflammatory cascade, which in turn aggravates Amyloid beta (Ab) deposition and tau-induced neurodegeneration, showing the relevance of this pro-inflammatory pathway as an early event in AD.

Mitochondrial DNA Repair in Neurodegenerative Diseases and Ageing.

Mitochondria are the only organelles, along with the nucleus, that have their own DNA. Mitochondrial DNA (mtDNA) is a double-stranded circular molecule of ~16.5 kbp that can exist in multiple copies within the organelle. Both strands are translated and encode for 22 tRNAs, 2 rRNAs, and 13 proteins. mtDNA molecules are anchored to the inner mitochondrial membrane and, in association with proteins, form a structure called nucleoid, which exerts a structural and protective function. Indeed, mitochondria have evolved mechanisms necessary to protect their DNA from chemical and physical lesions such as DNA repair pathways similar to those present in the nucleus. However, there are mitochondria-specific mechanisms such as rapid mtDNA turnover, fission, fusion, and mitophagy. Nevertheless, mtDNA mutations may be abundant in somatic tissue due mainly to the proximity of the mtDNA to the oxidative phosphorylation (OXPHOS) system and, consequently, to the reactive oxygen species (ROS) formed during ATP production. In this review, we summarise the most common types of mtDNA lesions and mitochondria repair mechanisms. The second part of the review focuses on the physiological role of mtDNA damage in ageing and the effect of mtDNA mutations in neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Considering the central role of mitochondria in maintaining cellular homeostasis, the analysis of mitochondrial function is a central point for developing personalised medicine.

AGE-TXNIP axis drives inflammation in Alzheimer's by targeting Aß to mitochondria in microglia.

Alzheimer's disease (AD) is the most common form of dementia characterized by progressive memory loss and cognitive decline. Although neuroinflammation and oxidative stress are well-recognized features of AD, their correlations with the early molecular events characterizing the pathology are not yet well clarified. Here, we characterize the role of RAGE-TXNIP axis in neuroinflammation in relation to amyloid-beta (Aß) burden in both in vivo and in vitro models. In the hippocampus of 5xFAD mice microglial activation, cytokine secretion, and glial fibrillary acidic protein-enhanced expression are paralleled with increased TXNIP expression. TXNIP silencing or its pharmacological inhibition prevents neuroinflammation in those mice. TXNIP is also associated with RAGE and Aß. In particular, RAGE-TXNIP axis is required for targeting Aß in mitochondria, leading to mitochondrial dysfunction and oxidative stress. Silencing of TXNIP or inhibition of RAGE activation reduces Aß transport from the cellular surface to mitochondria, restores mitochondrial functionality, and mitigates Aß toxicity. Furthermore, Aß shuttling into mitochondria promotes Drp1 activation and exacerbates mitochondrial dysfunction, which induces NLRP3 inflammasome activation, leading to secretion of IL-1ß and activation of the pyroptosis-associated protein Gasdermin D (GSDMD). Downregulation of RAGE-TXNIP axis inhibits Aß-induced mitochondria dysfunction, inflammation, and induction of GSDMD. Herein we unveil a new pathway driven by TXNIP that links the mitochondrial transport of Aß to the activation of Drp1 and the NLRP3 inflammasome, promoting the secretion of IL-1ß and the pyroptosis pathway associated with GSDMD cleavage. Altogether these data shed new light on a novel mechanism of action of RAGE-TXNIP axis in microglia, which is intertwined with Aß and ultimately causes mitochondria dysfunction and NLRP3 inflammasome cascade activation, suggesting TXNIP as a druggable target to be better deepened for AD.

DNA Repair Protein APE1 Degrades Dysfunctional Abasic mRNA in Mitochondria Affecting Oxidative Phosphorylation.

APE1 is a multifunctional protein which plays a central role in the maintenance of nuclear and mitochondrial genomes repairing DNA lesions caused by oxidative and alkylating agents. In addition, it works as a redox signaling protein regulating gene expression by interacting with many transcriptional factors. Apart from these canonical activities, recent studies have shown that APE1 is also enzymatically active on RNA molecules. The present study unveils for the first time a new role of the mitochondrial form of APE1 protein in the metabolism of RNA in mitochondria. Our data demonstrate that APE1 is associated with mitochondrial messenger RNA and exerts endoribonuclease activity on abasic sites. Loss of APE1 results in the accumulation of damaged mitochondrial mRNA species, determining impairment in protein translation and reduced expression of mitochondrial-encoded proteins, finally leading to less efficient mitochondrial respiration. Altogether, our data demonstrate that APE1 plays an active role in the degradation of the mitochondrial mRNA and has a profound impact on mitochondrial well-being.

Mitochondrial Oxidative Stress Induces Rapid Intermembrane Space/Matrix Translocation of Apurinic/Apyrimidinic Endonuclease 1 Protein through TIM23 Complex.

Mitochondria are essential cellular organelles that import the majority of proteins to sustain their function in cellular metabolism and homeostasis. Due to their role in oxidative phosphorylation, mitochondria are constantly affected by oxidative stress. Stability of mitochondrial DNA (mtDNA) is essential for mitochondrial physiology and cellular well-being and for this reason mtDNA lesions have to be rapidly recognized and repaired. Base excision repair (BER) is the main pathway responsible for repairing non-helix distorting base lesions both into the nucleus and in mitochondria. Apurinic/Apyrimidinic Endonuclease 1 (APE1) is a key component of BER pathway and the only protein that can recognize and process an abasic (AP) site. Comprehensions of the mechanisms regulating APE1 intracellular trafficking are still fragmentary. In this study we focused our attention on the mitochondrial form of APE1 protein and how oxidative stress induces its translocation to maintain mtDNA integrity. Our data proved that: (i) the rise of mitochondrial ROS determines a very rapid translocation of APE1 from the intermembrane space (IMS) into the matrix; and (ii) TIM23/PAM machinery complex is responsible for the matrix translocation of APE1. Moreover, our data support the hypothesis that the IMS, where the majority of APE1 resides, could represent a sort of storage site for the protein.

Mitochondrial apurinic/apyrimidinic endonuclease 1 enhances mtDNA repair contributing to cell proliferation and mitochondrial integrity in early stages of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the leading cause of primary liver cancers. Surveillance of individuals at specific risk of developing HCC, early diagnostic markers, and new therapeutic approaches are essential to obtain a reduction in disease-related mortality. Apurinic/apyrimidinic endonuclease 1 (APE1) expression levels and its cytoplasmic localization have been reported to correlate with a lower degree of differentiation and shorter survival rate. The aim of this study is to fully investigate, for the first time, the role of the mitochondrial form of APE1 in HCC. METHODS: As a study model, we analyzed samples from a cohort of patients diagnosed with HCC who underwent surgical resection. Mitochondrial APE1 content, expression levels of the mitochondrial import protein Mia40, and mtDNA damage of tumor tissue and distal non-tumor liver of each patient were analyzed. In parallel, we generated a stable HeLa clone for inducible silencing of endogenous APE1 and re-expression of the recombinant shRNA resistant mitochondrially targeted APE1 form (MTS-APE1). We evaluated mtDNA damage, cell growth, and mitochondrial respiration. RESULTS: APE1's cytoplasmic positivity in Grades 1 and 2 HCC patients showed a significantly higher expression of mitochondrial APE1, which accounted for lower levels of mtDNA damage observed in the tumor tissue with respect to the distal area. In the contrast, the cytoplasmic positivity in Grade 3 was not associated with APE1's mitochondrial accumulation even when accounting for the higher number of mtDNA lesions measured. Loss of APE1 expression negatively affected mitochondrial respiration, cell viability, and proliferation as well as levels of mtDNA damage. Remarkably, the phenotype was efficiently rescued in MTS-APE1 clone, where APE1 is present only within the mitochondrial matrix. CONCLUSIONS: Our study confirms the prominent role of the mitochondrial form of APE1 in the early stages of HCC development and the relevance of the non-nuclear fraction of APE1 in the disease progression. We have also confirmed overexpression of Mia40 and the role of the MIA pathway in the APE1 import process. Based on our data, inhibition of the APE1 transport by blocking the MIA pathway could represent a new therapeutic approach for reducing mitochondrial metabolism by preventing the efficient repair of mtDNA.

Autophagy and Inflammasome Activation in Dilated Cardiomyopathy.

BACKGROUND: The clinical outcome of patients affected by dilated cardiomyopathy (DCM) is heterogeneous, since its pathophysiology is only partially understood. Interleukin 1ß levels could predict the mortality and necessity of cardiac transplantation of DCM patients. OBJECTIVE: To investigate mechanisms triggering sterile inflammation in dilated cardiomyopathy (DCM). METHODS: Hearts explanted from 62 DCM patients were compared with 30 controls, employing immunohistochemistry, cellular and molecular biology, as well as metabolomics studies. RESULTS: Although misfolded protein accumulation and aggresome formation characterize DCM hearts, aggresomes failed to trigger the autophagy lysosomal pathway (ALP), with consequent accumulation of both p62SQSTM1 and dysfunctional mitochondria. In line, DCM hearts are characterized by accumulation of lipoperoxidation products and activation of both redox responsive pathways and inflammasome. Consistently with the fact that mTOR signaling may impair ALP, we observed, an increase in DCM activation, together with a reduction in the nuclear localization of Transcription Factor EB -TFEB- (a master regulator of lysosomal biogenesis). These alterations were coupled with metabolomic alterations, including accumulation of branched chain amino acids (BCAAs), known mTOR activators. Consistently, reduced levels of PP2Cm, a phosphatase that regulates the key catabolic step of BCAAs, coupled with increased levels of miR-22, a regulator of PP2Cm levels that triggers senescence, characterize DCM hearts. The same molecular defects were present in clinically relevant cells isolated from DCM hearts, but they could be reverted by downregulating miR-22. CONCLUSION: We identified, in human DCM, a complex series of events whose key players are miR-22, PP2Cm, BCAA, mTOR, and ALP, linking loss of proteostasis with inflammasome activation. These potential therapeutic targets deserve to be further investigated.

Transcription, Processing, and Decay of Mitochondrial RNA in Health and Disease.

Although the large majority of mitochondrial proteins are nuclear encoded, for their correct functioning mitochondria require the expression of 13 proteins, two rRNA, and 22 tRNA codified by mitochondrial DNA (mtDNA). Once transcribed, mitochondrial RNA (mtRNA) is processed, mito-ribosomes are assembled, and mtDNA-encoded proteins belonging to the respiratory chain are synthesized. These processes require the coordinated spatio-temporal action of several enzymes, and many different factors are involved in the regulation and control of protein synthesis and in the stability and turnover of mitochondrial RNA. In this review, we describe the essential steps of mitochondrial RNA synthesis, maturation, and degradation, the factors controlling these processes, and how the alteration of these processes is associated with human pathologies.

[Letter to the Editor] Isolation of mitochondria is necessary for precise quantification of mitochondrial DNA damage in human carcinoma samples.

Address correspondence to Carlo Vascotto, Department of Medical and Biological Sciences, University of Udine, Udine, 33100, Italy. E-mail: carlo.vascotto@uniud.it.

Identification of tumorigenesis-related mRNAs associated with RNA-binding protein HuR in thyroid cancer cells.

RNA binding proteins (RBPs) play a central role in cell physiology and pathology. Among them, HuR is a nuclear RBP, which shuttles to the cytoplasm to allow its RNA targets processing. HuR over-expression and delocalization are often associated to cell transformation. Numerous cancers display increased HuR protein levels and its high cytoplasmic levels has been associated with a worse prognosis.In our study, we first evaluated HuR expression in normal and cancer thyroid tissues and then evaluated its function in thyroid cell lines. HuR is over-expressed in all thyroid tumor tissues; high cytoplasmic levels are detected in all thyroid carcinomas. HuR silencing decreased cell viability and determined apoptotic cell death, in a non-tumorigenic (Nthy-ori-3.1) and a tumorigenic (BCPAP) thyroid cell line. Global transcriptome analysis indicated that HuR silencing, though having similar biological effects, induces distinct gene expression modifications in the two cell lines. By using the RIP-seq approach, the HuR-bound RNA profiles of different thyroid cell lines were evaluated. We show that in distinct cell lines HuR-bound RNA profiles are different. A set of 114 HuR-bound RNAs distinguishing tumorigenic cell lines from the non-tumorigenic one was identified.Altogether, our data indicate that HuR plays a role in thyroid tumorigenesis. Moreover, our findings are a proof of concept that RBP targets differ between cells with the same origin but with distinct biological behavior.

Inhibitors of the apurinic/apyrimidinic endonuclease 1 (APE1)/nucleophosmin (NPM1) interaction that display anti-tumor properties.

The apurinic/apyrimidinic endonuclease 1 (APE1) is a protein central to the base excision DNA repair pathway and operates in the modulation of gene expression through redox-dependent and independent mechanisms. Aberrant expression and localization of APE1 in tumors are recurrent hallmarks of aggressiveness and resistance to therapy. We identified and characterized the molecular association between APE1 and nucleophosmin (NPM1), a multifunctional protein involved in the preservation of genome stability and rRNA maturation. This protein-protein interaction modulates subcellular localization and endonuclease activity of APE1. Moreover, we reported a correlation between APE1 and NPM1 expression levels in ovarian cancer, with NPM1 overexpression being a marker of poor prognosis. These observations suggest that tumors that display an augmented APE1/NPM1 association may exhibit increased aggressiveness and resistance. Therefore, targeting the APE1/NPM1 interaction might represent an innovative strategy for the development of anticancer drugs, as tumor cells relying on higher levels of APE1 and NPM1 for proliferation and survival may be more sensitive than untransformed cells. We set up a chemiluminescence-based high-throughput screening assay in order to find small molecules able to interfere with the APE1/NPM1 interaction. This screening led to the identification of a set of bioactive compounds that impair the APE1/NPM1 association in living cells. Interestingly, some of these molecules display anti-proliferative activity and sensitize cells to therapeutically relevant genotoxins. Given the prognostic significance of APE1 and NPM1, these compounds might prove effective in the treatment of tumors that show abundant levels of both proteins, such as ovarian or hepatic carcinomas.

Transcriptional Up-Regulation of APE1/Ref-1 in Hepatic Tumor: Role in Hepatocytes Resistance to Oxidative Stress and Apoptosis.

OBJECTIVE: Human Hepatocellular Carcinoma (HCC) is the fifth most frequent neoplasm worldwide and the most serious complication of long-standing chronic liver diseases (CLD). Its development is associated with chronic inflammation and sustained oxidative stress. Deregulation of apurinic apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1), a master regulator of cellular response to oxidative stress, has been associated with poor prognosis in several cancers including HCC. DESIGN: In the present study we investigated the APE1/Ref-1 mRNA levels in cirrhotic and HCC tissues obtained during HCC resection. The possible protective role of APE1/Ref-1 against oxidative stress and apoptosis was evaluated in vitro in immortalized human hepatocytes (IHH) over-expressing APE1/Ref-1. RESULTS: APE1/Ref-1 was up-regulated in HCC, regulation occurring at the transcriptional level. APE1/Ref-1 mRNA content increased with the progression of liver disease with the transcriptional up-regulation present in cirrhosis significantly increased in HCC. The up-regulation was higher in the less differentiated cancers. In vitro, over-expression of APE1/Ref-1 in normal hepatocytes conferred cell protection against oxidative stress and it was associated with BAX inhibition and escape from apoptosis. CONCLUSION: APE1/Ref-1 is up-regulated in HCC and this over-expression correlates with cancer aggressiveness. The up-regulation occurs at the transcriptional level and it is present in the earliest phases of hepatocarcinogenesis. The APE-1/Ref-1 over-expression is associated with hepatocyte survival and inhibits BAX activation and apoptosis. These data suggest a possible role of APE1/Ref-1 over-expression both in hepatocyte survival and HCC development calling attention to this molecule as a promising marker for HCC diagnosis and treatment.

Mitochondrial translocation of APE1 relies on the MIA pathway.

APE1 is a multifunctional protein with a fundamental role in repairing nuclear and mitochondrial DNA lesions caused by oxidative and alkylating agents. Unfortunately, comprehensions of the mechanisms regulating APE1 intracellular trafficking are still fragmentary and contrasting. Recent data demonstrate that APE1 interacts with the mitochondrial import and assembly protein Mia40 suggesting the involvement of a redox-assisted mechanism, dependent on the disulfide transfer system, to be responsible of APE1 trafficking into the mitochondria. The MIA pathway is an import machinery that uses a redox system for cysteine enriched proteins to drive them in this compartment. It is composed by two main proteins: Mia40 is the oxidoreductase that catalyzes the formation of the disulfide bonds in the substrate, while ALR reoxidizes Mia40 after the import. In this study, we demonstrated that: (i) APE1 and Mia40 interact through disulfide bond formation; and (ii) Mia40 expression levels directly affect APE1's mitochondrial translocation and, consequently, play a role in the maintenance of mitochondrial DNA integrity. In summary, our data strongly support the hypothesis of a redox-assisted mechanism, dependent on Mia40, in controlling APE1 translocation into the mitochondrial inner membrane space and thus highlight the role of this protein transport pathway in the maintenance of mitochondrial DNA stability and cell survival.

Ex vivo molecular rejuvenation improves the therapeutic activity of senescent human cardiac stem cells in a mouse model of myocardial infarction.

Cardiac stem cells (CSC) from explanted decompensated hearts (E-CSC) are, with respect to those obtained from healthy donors (D-CSC), senescent and functionally impaired. We aimed to identify alterations in signaling pathways that are associated with CSC senescence. Additionally, we investigated if pharmacological modulation of altered pathways can reduce CSC senescence in vitro and enhance their reparative ability in vivo. Measurement of secreted factors showed that E-CSC release larger amounts of proinflammatory cytokine IL1ß compared with D-CSC. Using blocking antibodies, we verified that IL1ß hampers the paracrine protective action of E-CSC on cardiomyocyte viability. IL1ß acts intracranially inducing IKKß signaling, a mechanism that via nuclear factor-κB upregulates the expression of IL1ß itself. Moreover, E-CSC show reduced levels of AMP protein kinase (AMPK) activating phosphorylation. This latter event, together with enhanced IKKß signaling, increases TORC1 activity, thereby impairing the autophagic flux and inhibiting the phosphorylation of Akt and cAMP response element-binding protein. The combined use of rapamycin and resveratrol enhanced AMPK, thereby restoring downstream signaling and reducing IL1ß secretion. These molecular corrections reduced E-CSC senescence, re-establishing their protective activity on cardiomyocytes. Moreover ex vivo treatment with rapamycin and resveratrol improved E-CSC capacity to induce cardiac repair upon injection in the mouse infarcted heart, leading to reduced cardiomyocyte senescence and apoptosis and increased abundance of endogenous c-Kit(+) CSC in the peri-infarct area. Molecular rejuvenation of patient-derived CSC by short pharmacologic conditioning boosts their in vivo reparative abilities. This approach might prove useful for refinement of CSC-based therapies.

The redox function of APE1 is involved in the differentiation process of stem cells toward a neuronal cell fate.

UNLABELLED: Low-to-moderate levels of reactive oxygen species (ROS) govern different steps of neurogenesis via molecular pathways that have been decrypted only partially. Although it has been postulated that redox-sensitive molecules are involved in neuronal differentiation, the molecular bases for this process have not been elucidated yet. The aim of this work was therefore to study the role played by the redox-sensitive, multifunctional protein APE1/Ref-1 (APE1) in the differentiation process of human adipose tissue-derived multipotent adult stem cells (hAT-MASC) and embryonic carcinoma stem cells (EC) towards a neuronal phenotype. METHODS AND RESULTS: Applying a definite protocol, hAT-MASC can adopt a neural fate. During this maturation process, differentiating cells significantly increase their intracellular Reactive Oxygen Species (ROS) levels and increase the APE1 nuclear fraction bound to chromatin. This latter event is paralleled by the increase of nuclear NF-κB, a transcription factor regulated by APE1 in a redox-dependent fashion. Importantly, the addition of the antioxidant N-acetyl cysteine (NAC) to the differentiation medium partially prevents the nuclear accumulation of APE1, increasing the neuronal differentiation of hAT-MASC. To investigate the involvement of APE1 in the differentiation process, we employed E3330, a specific inhibitor of the APE1 redox function. The addition of E3330, either to the neurogenic embryonic carcinoma cell line NT2-D1or to hAT-MASC, increases the differentiation of stem cells towards a neural phenotype, biasing the differentiation towards specific subtypes, such as dopaminergic cells. In conclusion, during the differentiation process of stem cells towards a neuroectodermic phenotype, APE1 is recruited, in a ROS-dependent manner, to the chromatin. This event is associated with an inhibitory effect of APE1 on neurogenesis that may be reversed by E3330. Therefore, E3330 may be employed both to boost neural differentiation and to bias the differentiation potential of stem cells towards specific neuronal subtypes. These findings provide a molecular basis for the redox-mediated hypothesis of neuronal differentiation program.

Expression and prognostic significance of APE1/Ref-1 and NPM1 proteins in high-grade ovarian serous cancer.

OBJECTIVES: To correlate the expression profile of human apurinic endonuclease/redox factor 1 (APE1/Ref-1) with that of nucleolar/nucleoplasmic protein nucleophosmin 1 (NPM1) in association with the aggressiveness and progression of high-grade ovarian serous cancer. METHODS: Retrospective study analyzing a tissue microarray of 73 women affected by high-grade ovarian serous cancer. Protein expression was assessed by immunohistochemistry on primary tumor masses and synchronous peritoneal metastases if present. RESULTS: APE1/Ref-1 and NPM1 showed a significant correlation in ovarian serous cancer. Patients with a poorer outcome showed a significant overexpression of nuclear NPM1 protein. A Cox proportional hazards multivariate regression model revealed NPM1 expression to be independently significant for overall survival in high-grade ovarian serous cancers after correcting for stage, age, cytoreduction completeness, and platinum resistance. CONCLUSIONS: APE1/Ref-1 interacts with NPM1 to control the DNA damage repair system, and it is likely that this interaction plays a defining role in high-grade ovarian serous carcinoma. A high NPM1 immunohistochemical expression was independently correlated with a shorter survival period and thus appears to be an important prognostic factor.

Osteoblastic cell secretome: a novel role for progranulin during risedronate treatment.

It is well established that osteoblasts, the key cells involved in bone formation during development and in adult life, secrete a number of glycoproteins harboring autocrine and paracrine functions. Thus, investigating the osteoblastic secretome could yield important information for the pathophysiology of bone. In the present study, we characterized for the first time the secretome of human Hobit osteoblastic cells. We discovered that the secretome comprised 89 protein species including the powerful growth factor progranulin. Recombinant human progranulin (6nM) induced phosphorylation of mitogen-activated protein kinase in both Hobit and osteocytic cells and induced cell proliferation and survival. Notably, risedronate, a nitrogen-containing bisphosphonate widely used in the treatment of osteoporosis, induced the expression and secretion of progranulin in the Hobit secretome. In addition, our proteomic study of the Hobit secretome revealed that risedronate induced the expression of ERp57, HSP60 and HSC70, three proteins already shown to be associated with the prevention of bone loss in osteoporosis. Collectively, our findings unveil novel targets of risedronate-evoked biological effects on osteoblast-like cells and further our understanding of the mechanisms of action of this currently used compound.

SIRT1 gene expression upon genotoxic damage is regulated by APE1 through nCaRE-promoter elements.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein contributing to genome stability via repair of DNA lesions via the base excision repair pathway. It also plays a role in gene expression regulation and RNA metabolism. Another, poorly characterized function is its ability to bind to negative calcium responsive elements (nCaRE) of some gene promoters. The presence of many functional nCaRE sequences regulating gene transcription can be envisioned, given their conservation within ALU repeats. To look for functional nCaRE sequences within the human genome, we performed bioinformatic analyses and identified 57 genes potentially regulated by APE1. We focused on sirtuin-1 (SIRT1) deacetylase due to its involvement in cell stress, including senescence, apoptosis, and tumorigenesis, and its role in the deacetylation of APE1 after genotoxic stress. The human SIRT1 promoter presents two nCaRE elements stably bound by APE1 through its N-terminus. We demonstrate that APE1 is part of a multiprotein complex including hOGG1, Ku70, and RNA Pol II, which is recruited on SIRT1 promoter to regulate SIRT1 gene functions during early response to oxidative stress. These findings provide new insights into the role of nCaRE sequences in the transcriptional regulation of mammalian genes.