Retraction Note: NF-κB activation impairs somatic cell reprogramming in ageing.

We, the authors, are retracting this Article due to issues that have come to our attention regarding data availability, data description and figure assembly. Specifically, original numerical data are not available for the majority of the graphs presented in the paper. Although original data were available for most EMSA and immunoblot experiments, those corresponding to the published EMSA data of Supplementary Fig. 8a, the independent replicate immunoblots of Fig. 8b and Supplementary Fig. 1e, and the independent replicate EMSA data of Supplementary Figs 6e, 8b, 8c and 8d, are unavailable. Mistakes were detected in the presentation of Figs 3c, 4i and Supplementary Figs 6a, 8a, 8d, 9, and in some cases the ß-actin immunoblots were erroneously described in the figure legends as loading controls, rather than as sample processing controls that were run on separate gels. Although we, the authors, believe that the key findings of the paper are still valid, given the issues with data availability we have concluded that the most appropriate course of action is to retract the Article. We deeply regret these errors and apologize to the scientific community for any confusion this publication may have caused. All authors agree with the retraction.

Haematopoietic stem and progenitor cells from human pluripotent stem cells.

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders.

Immune and inflammatory responses to DNA damage in cancer and aging.

Genome instability is a hallmark of both cancer and aging processes. Beyond cell-autonomous responses, it is known that DNA damage also elicits systemic mechanisms aimed at favoring survival and damaged cells clearance. Among these mechanisms, immune activation and NF-κB-mediated inflammation play central roles in organismal control of DNA damage. We focus herein on the different experimental evidences that have allowed gaining mechanistic insight about this relationship. We also describe the functional consequences of defective immune function in cancer development and age-related alterations. Finally, we discuss different intervention strategies based on enhancing immunity or on the modulation of the inflammatory response to improve organism homeostasis in cancer and aging.

Hallmarks of progeroid syndromes: lessons from mice and reprogrammed cells.

Ageing is a process that inevitably affects most living organisms and involves the accumulation of macromolecular damage, genomic instability and loss of heterochromatin. Together, these alterations lead to a decline in stem cell function and to a reduced capability to regenerate tissue. In recent years, several genetic pathways and biochemical mechanisms that contribute to physiological ageing have been described, but further research is needed to better characterize this complex biological process. Because premature ageing (progeroid) syndromes, including progeria, mimic many of the characteristics of human ageing, research into these conditions has proven to be very useful not only to identify the underlying causal mechanisms and identify treatments for these pathologies, but also for the study of physiological ageing. In this Review, we summarize the main cellular and animal models used in progeria research, with an emphasis on patient-derived induced pluripotent stem cell models, and define a series of molecular and cellular hallmarks that characterize progeroid syndromes and parallel physiological ageing. Finally, we describe the therapeutic strategies being investigated for the treatment of progeroid syndromes, and their main limitations.

iPSCs: On the Road to Reprogramming Aging.

Aging is characterized by irreversible loss of physiological integrity, often accompanied by an organism's loss of function and increased vulnerability to death. Defects in the mechanisms preserving cellular homeostasis over time may give rise to accelerated aging. Somatic cell reprogramming of aged cells can be associated with rejuvenation, erasing certain age-associated features, and illustrating the reversibility potential of aging. Here, we focus on recent advances in the generation of human induced pluripotent stem cells from progeroid syndromes and late-onset diseases such as Alzheimer's or Parkinson's. These cellular models have contributed to a better understanding of such pathologies, as well as to the development of novel therapeutic approaches. We also discuss different strategies to identify and target age-associated reprogramming barriers to facilitate the treatment of age-related disorders.

Novel LMNA mutations cause an aggressive atypical neonatal progeria without progerin accumulation.

BACKGROUND: Progeroid syndromes are genetic disorders that recapitulate some phenotypes of physiological ageing. Classical progerias, such as Hutchinson-Gilford progeria syndrome (HGPS), are generally caused by mutations in LMNA leading to accumulation of the toxic protein progerin and consequently, to nuclear envelope alterations. In this work, we describe a novel phenotypic feature of the progeria spectrum affecting three unrelated newborns and identify its genetic cause. METHODS AND RESULTS: Patients reported herein present an extremely homogeneous phenotype that somewhat recapitulates those of patients with HGPS and mandibuloacral dysplasia. However, pathological signs appear earlier, are more aggressive and present distinctive features including episodes of severe upper airway obstruction. Exome and Sanger sequencing allowed the identification of heterozygous de novo c.163G>A, p.E55K and c.164A>G, p.E55G mutations in LMNA as the alterations responsible for this disorder. Functional analyses demonstrated that fibroblasts from these patients suffer important dysfunctions in nuclear lamina, which generate profound nuclear envelope abnormalities but without progerin accumulation. These nuclear alterations found in patients' dermal fibroblasts were also induced by ectopic expression of the corresponding site-specific LMNA mutants in control human fibroblasts. CONCLUSIONS: Our results demonstrate the causal role of p.E55K and p.E55G lamin A mutations in a disorder which manifests novel phenotypic features of the progeria spectrum characterised by neonatal presentation and aggressive clinical evolution, despite being caused by lamin A/C missense mutations with effective prelamin A processing.

MMP-25 Metalloprotease Regulates Innate Immune Response through NF-κB Signaling.

Matrix metalloproteases (MMPs) regulate innate immunity acting over proinflammatory cytokines, chemokines, and other immune-related proteins. MMP-25 (membrane-type 6-MMP) is a membrane-bound enzyme predominantly expressed in leukocytes whose biological function has remained largely unknown. We have generated Mmp25-deficient mice to elucidate the in vivo function of this protease. These mutant mice are viable and fertile and do not show any spontaneous phenotype. However, Mmp25-null mice exhibit a defective innate immune response characterized by low sensitivity to bacterial LPS, hypergammaglobulinemia, and reduced secretion of proinflammatory molecules. Moreover, these immune defects can be tracked to a defective NF-κB activation observed in Mmp25-deficient leukocytes. Globally, our findings provide new mechanistic insights into innate immunity through the activity of MMP-25, suggesting that this proteinase could be a potential therapeutic target for immune-related diseases.

Loss of the proteostasis factor AIRAPL causes myeloid transformation by deregulating IGF-1 signaling.

AIRAPL (arsenite-inducible RNA-associated protein-like) is an evolutionarily conserved regulator of cellular proteostasis linked to longevity in nematodes, but its biological function in mammals is unknown. We show herein that AIRAPL-deficient mice develop a fully-penetrant myeloproliferative neoplastic process. Proteomic analysis of AIRAPL-deficient mice revealed that this protein exerts its antineoplastic function through the regulation of the insulin/insulin-like growth factor 1 (IGF-1) signaling pathway. We demonstrate that AIRAPL interacts with newly synthesized insulin-related growth factor-1 receptor (IGF1R) polypeptides, promoting their ubiquitination and proteasome-mediated degradation. Accordingly, genetic and pharmacological IGF1R inhibitory strategies prevent the hematological disease found in AIRAPL-deficient mice as well as that in mice carrying the Jak2(V617F) mutation, thereby demonstrating the causal involvement of this pathway in the pathogenesis of myeloproliferative neoplasms. Consistent with its proposed role as a tumor suppressor of myeloid transformation, AIRAPL expression is widely abrogated in human myeloproliferative disorders. Collectively, these findings support the oncogenic relevance of proteostasis deregulation in hematopoietic cells, and they unveil novel therapeutic targets for these frequent hematological neoplasias.

NF-κB activation impairs somatic cell reprogramming in ageing.

Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo progeria syndrome and Hutchinson-Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.

Loss of MT1-MMP causes cell senescence and nuclear defects which can be reversed by retinoic acid.

MT1-MMP (MMP14) is a collagenolytic enzyme located at the cell surface and implicated in extracellular matrix (ECM) remodeling. Mmp14(-/-) mice present dwarfism, bone abnormalities, and premature death. We demonstrate herein that the loss of MT1-MMP also causes cardiac defects and severe metabolic changes, and alters the cytoskeleton and the nuclear lamina structure. Moreover, the absence of MT1-MMP induces a senescent phenotype characterized by up-regulation of p16(INK4a) and p21(CIP1/WAF) (1), increased activity of senescence-associated ß-galactosidase, generation of a senescence-associated secretory phenotype, and somatotroph axis alterations. Consistent with the role of retinoic acid signaling in nuclear lamina stabilization, treatment of Mmp14(-/-) mice with all-trans retinoic acid reversed the nuclear lamina alterations, partially rescued the cell senescence phenotypes, ameliorated the pathological defects in bone, skin, and heart, and extended their life span. These results demonstrate that nuclear architecture and cell senescence can be modulated by a membrane protease, in a process involving the ECM as a key regulator of nuclear stiffness under cell stress conditions.

Effects of azacitidine on matrix metalloproteinase-9 in acute myeloid leukemia and myelodysplasia.

Matrix metalloprotease-9 (MMP9) plays a critical role in acute myeloid leukemia (AML) by increasing the invasive properties of malignant myeloblasts. The role of this enzyme in high-risk myelodysplastic diseases (MDS) and the effect of azacitidine on its expression in MDS and AML have not been studied in detail. In this work, we have analyzed the effect of different concentrations of azacitidine in two well-established, MDS-derived, acute myeloid leukemic cell lines: MOLM-13 and SKM-1. We have demonstrated that 1 µmol/L azacitidine decreases MMP9 DNA methylation levels and that this is correlated with a significant increase in messenger RNA expression in both cell lines. Surprisingly, changes in protein levels were minor. This paradoxic effect is explained by the drug-dependent induction of apoptosis that reduces the amount of active secreting cells. A balance between induced expression and apoptosis was established at an azacitidine concentration of 0.2 µmol/L in MOLM-13 cells. This dose significantly increased the invasive capacity of viable cells, as measured in the Matrigel assay. To evaluate the clinical relevance of this observation, we have examined the effect of azacitidine on MMP9 expression in bone marrow from five patients with MDS, with the finding that this drug significantly increased MMP9 protein levels in all analyzed patients after six cycles of treatment. Based on these results, we conclude that azacitidine increases MMP9 expression and may enhance invasiveness in vitro. Because all five patients relapsed, these findings might explain, at least partially, the clinical failure of the drug and the progression to a more aggressive disease.

Nuclear lamina defects cause ATM-dependent NF-κB activation and link accelerated aging to a systemic inflammatory response.

Alterations in the architecture and dynamics of the nuclear lamina have a causal role in normal and accelerated aging through both cell-autonomous and systemic mechanisms. However, the precise nature of the molecular cues involved in this process remains incompletely defined. Here we report that the accumulation of prelamin A isoforms at the nuclear lamina triggers an ATM- and NEMO-dependent signaling pathway that leads to NF-κB activation and secretion of high levels of proinflammatory cytokines in two different mouse models of accelerated aging (Zmpste24(-/-) and Lmna(G609G/G609G) mice). Causal involvement of NF-κB in accelerated aging was demonstrated by the fact that both genetic and pharmacological inhibition of NF-κB signaling prevents age-associated features in these animal models, significantly extending their longevity. Our findings provide in vivo proof of principle for the feasibility of pharmacological modulation of the NF-κB pathway to slow down the progression of physiological and pathological aging.

Analysis of the disintegrin-metalloproteinases family reveals ADAM29 and ADAM7 are often mutated in melanoma.

We performed a mutational analysis of the 19 disintegrin-metalloproteinases (ADAMs) genes in human cutaneous metastatic melanoma and identified eight to be somatically mutated in 79 samples, affecting 34% of the melanoma tumors analyzed. Functional analysis of the two frequently mutated ADAM genes, ADAM29 and ADAM7 demonstrated that the mutations affect adhesion of melanoma cells to specific extracellular matrix proteins and in some cases increase their migration ability. This suggests that mutated ADAM genes could play a role in melanoma progression.

Sexual autophagic differences in the androgen-dependent flank organ of Syrian hamsters.

The flank organ of the Syrian hamster shows a biodynamic response to androgenic stimulation and is, therefore, a suitable model for the study of androgenic effects on hair and sebaceous glands. This organ is susceptible to programmed cell death (PCD), a prominent feature associated with sexual organ adjustment. In the present report, the type of PCD (apoptosis or autophagy) exhibited by this organ was evaluated. Caspase-3 activity, indicative of apoptosis, was not detectable in flank organ homogenates. Furthermore, cytokeratins, which are normally degraded during apoptosis, remained intact. On the other hand, Western blotting of Beclin 1 and light chain 3-II, both important autophagy markers, revealed autophagic processes in the flank organ in both sexes, especially in females. Cathepsin D activity, higher in males than in females, and procathepsin D expression were also consistent with autophagy and not apoptosis. Taken together, these data indicate that macroautophagy, and not apoptosis, is the main mechanism by which the flank organ responds to androgen. This is the first direct evidence establishing the relationship between autophagy and morphological changes in androgen-dependent organs.

Melatonin alters cell death processes in response to age-related oxidative stress in the brain of senescence-accelerated mice.

We studied the effect of age and melatonin on cell death processes in brain aging. Senescence-accelerated prone mice 8 (SAMP8) and senescence-accelerated resistant mice (SAMR1) at 5 and 10 months of age were used as models of the study. Melatonin (10 mg/kg) or its vehicle (ethanol at 0.066%) was administered in the drinking water from 1 to 9 months of age. Neurodegeneration, previously shown in the aged brain of SAMP8 and SAMR1 at 10 months of age, may be due to a drop in age-related proteolytic activities (cathepsin D, calpains, and caspase-3). Likewise, lack of apoptotic and macroautophagic processes were found, without apparent modification by melatonin. However, the caspase-independent cell death, owing to high p53 and apoptosis-inducing factor (AIF) levels, might be an alternative pathway of cell death in the aged brain. The main effects of melatonin treatment were observed in the aged SAMR1 mice; in this strain we observed a marked increase in antioxidant activity (catalase and superoxide dismutase). Likewise, a key antioxidant role of apoptosis-related proteins, Bcl-2 and AIF, was suggested in the aged brain of SAM mice, which was clearly influenced by melatonin. Moreover, the age-related increase of lysosomal activity of cathepsin B and a lysosomal membrane-associated protein 2 supports the possibility of the maintenance of lysosomal viability in addition to age-related impairments of the proteolytic or macroautophagic activities. The effectiveness of melatonin against the oxidative stress-related impairments and apoptosis during the aging process is, once more, corroborated in this article.

Favorable effects of a prolonged treatment with melatonin on the level of oxidative damage and neurodegeneration in senescence-accelerated mice.

Senescence-accelerated mice (SAMP8) and senescence-accelerated resistant mice (SAMR1) were studied at 5 and 10 months of age, respectively. In the animals, neurodegenerative processes and how they were influenced by melatonin were examined. Melatonin (10 mg/kg) or vehicle (ethanol at 0.066%) treatments were administrated from the age of 1 to 9 months in the drinking water. Differences in the neurodegenerative markers examined were found between the two strains with a more damaged protein, phosphorylated Tau at Ser392, increased neurofibrillary tangles (NT) and higher alpha-synuclein expression in SAMP8 versus SAMR1 mice overall, when the mice were 10 months of age. Changes in density of receptors and oxidative stress-related signaling with age were found in the brains of SAM strains at 10 months as shown by a marked decrease in the level of MT-1 melatonin receptor and retinoic acid receptor-related orphan receptor (ROR)-alpha1. This diminution was earlier and more pronounced in SAMP8 mice. Likewise, the levels of nuclear factor-kappa B (NF-kB) transcriptional factor were higher in SAMP8 mice compared with SAMR1 mice regardless of age confirming the direct role of oxidative stress in the aging process. Treatment with melatonin in SAMP8 and SAMR1 mice reduced the neurodegenerative changes with an increase of ROR-alpha1 levels without an apparent influence in the levels of MT-1 receptor. However, different melatonin effects on NF-kB signaling were observed suggesting that NF-kB could trigger inflammatory processes in a different way, being SAM strain-dependent and associated with age-related oxidative stress levels. The effectiveness of melatonin in improving age-related neural impairments is corroborated.