Lucia Micutkova

miR-17, miR-19b, miR-20a, and miR-106a are down-regulated in human aging.

Aging is a multifactorial process where deterioration of body functions is driven by stochastic damage while counteracted by distinct genetically encoded repair systems. To better understand the genetic component of aging, many studies have addressed the gene and protein expression profiles of various aging model systems engaging different organisms from yeast to human. The recently identified small non‐coding miRNAs are potent post‐transcriptional regulators that can modify the expression of up to several hundred target genes per single miRNA, similar to transcription factors. Increasing evidence shows that miRNAs contribute to the regulation of most if not all important physiological processes, including aging. However, so far the contribution of miRNAs to age‐related and senescence‐related changes in gene expression remains elusive. To address this question, we have selected four replicative cell aging models including endothelial cells, replicated CD8+ T cells, renal proximal tubular epithelial cells, and skin fibroblasts. Further included were three organismal aging models including foreskin, mesenchymal stem cells, and CD8+ T cell populations from old and young donors. Using locked nucleic acid‐based miRNA microarrays, we identified four commonly regulated miRNAs, miR‐17 down‐regulated in all seven; miR‐19b and miR‐20a, down‐regulated in six models; and miR‐106a down‐regulated in five models. Decrease in these miRNAs correlated with increased transcript levels of some established target genes, especially the cdk inhibitor p21/CDKN1A. These results establish miRNAs as novel markers of cell aging in humans.

Secreted microvesicular miR-31 inhibits osteogenic differentiation of mesenchymal stem cells

Damage to cells and tissues is one of the driving forces of aging and age‐related diseases. Various repair systems are in place to counteract this functional decline. In particular, the property of adult stem cells to self‐renew and differentiate is essential for tissue homeostasis and regeneration. However, their functionality declines with age (Rando, 2006). One organ that is notably affected by the reduced differentiation capacity of stem cells with age is the skeleton. Here, we found that circulating microvesicles impact on the osteogenic differentiation capacity of mesenchymal stem cells in a donor‐age‐dependent way. While searching for factors mediating the inhibitory effect of elderly derived microvesicles on osteogenesis, we identified miR‐31 as a crucial component. We demonstrated that miR‐31 is present at elevated levels in the plasma of elderly and of osteoporosis patients. As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR‐31 is secreted within senescent cell‐derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation by knocking down its target Frizzled‐3. Therefore, we suggest that microvesicular miR‐31 in the plasma of elderly might play a role in the pathogenesis of age‐related impaired bone formation and that miR‐31 might be a valuable plasma‐based biomarker for aging and for a systemic environment that does not favor cell‐based therapies whenever osteogenesis is a limiting factor.

Identification of evolutionarily conserved genetic regulators of cellular aging

To identify new genetic regulators of cellular aging and senescence, we performed genome‐wide comparative RNA profiling with selected human cellular model systems, reflecting replicative senescence, stress‐induced premature senescence, and distinct other forms of cellular aging. Gene expression profiles were measured, analyzed, and entered into a newly generated database referred to as the GiSAO database. Bioinformatic analysis revealed a set of new candidate genes, conserved across the majority of the cellular aging models, which were so far not associated with cellular aging, and highlighted several new pathways that potentially play a role in cellular aging. Several candidate genes obtained through this analysis have been confirmed by functional experiments, thereby validating the experimental approach. The effect of genetic deletion on chronological lifespan in yeast was assessed for 93 genes where (i) functional homologues were found in the yeast genome and (ii) the deletion strain was viable. We identified several genes whose deletion led to significant changes of chronological lifespan in yeast, featuring both lifespan shortening and lifespan extension. In conclusion, an unbiased screen across species uncovered several so far unrecognized molecular pathways for cellular aging that are conserved in evolution.

Identification of microRNA-mRNA functional interactions in UVB-induced senescence of human diploid fibroblasts

BackgroundCellular senescence can be induced by a variety of extrinsic stimuli, and sustained exposure to sunlight is a key factor in photoaging of the skin. Accordingly, irradiation of skin fibroblasts by UVB light triggers cellular senescence, which is thought to contribute to extrinsic skin aging, although molecular mechanisms are incompletely understood. Here, we addressed molecular mechanisms underlying UVB induced senescence of human diploid fibroblasts.ResultsWe observed a parallel activation of the p53/p21WAF1 and p16INK4a/pRb pathways. Using genome-wide transcriptome analysis, we identified a transcriptional signature of UVB-induced senescence that was conserved in three independent strains of human diploid fibroblasts (HDF) from skin. In parallel, a comprehensive screen for microRNAs regulated during UVB-induced senescence was performed which identified five microRNAs that are significantly regulated during the process. Bioinformatic analysis of miRNA-mRNA networks was performed to identify new functional mRNA targets with high confidence for miR-15a, miR-20a, miR-20b, miR-93, and miR-101. Already known targets of these miRNAs were identified in each case, validating the approach. Several new targets were identified for all of these miRNAs, with the potential to provide new insight in the process of UVB-induced senescence at a genome-wide level. Subsequent analysis was focused on miR-101 and its putative target gene Ezh2. We confirmed that Ezh2 is regulated by miR-101 in human fibroblasts, and found that both overexpression of miR-101 and downregulation of Ezh2 independently induce senescence in the absence of UVB irradiation. However, the downregulation of miR-101 was not sufficient to block the phenotype of UVB-induced senescence, suggesting that other UVB-induced processes induce the senescence response in a pathway redundant with upregulation of miR-101.ConclusionWe performed a comprehensive screen for UVB-regulated microRNAs in human diploid fibroblasts, and identified a network of miRNA-mRNA interactions mediating UVB-induced senescence. In addition, miR-101 and Ezh2 were identified as key players in UVB-induced senescence of HDF.

Role of endonuclease G in senescence-associated cell death of human endothelial cells

Mitotic cells in culture show a limited replicative potential and after extended subculturing undergo a terminal growth arrest termed cellular senescence. When cells reach the senescent phenotype, this is accompanied by a significant change in the cellular phenotype and massive changes in gene expression, including the upregulation of secreted factors. In human fibroblasts, senescent cells also acquire resistance to apoptosis. In contrary, in human endothelial cells, both replicative and stress-induced premature senescence is accompanied by increased cell death; however mechanisms of cell death are poorly explored. In this communication, we addressed the role of endonuclease G (EndoG), a mitochondrial mediator of caspase-independent cell death, in senescence-associated cell death of human endothelial cells. Using immunofluorescence microscopy, we found, that EndoG is localized in the mitochondria in young cells, but relocalizes to the nucleus upon senescence. When EndoG gene expression was downregulated by lentiviral shRNA vectors, we found a significant reduction in the replicative life span and a corresponding increase in cell death. We also observed a slight shift in the cell death phenotype from necrosis to apoptosis. Together these observations suggest an important role of EndoG in the senescence program of human endothelial cells.

Role of Insulin-Like Growth Factor Binding Protein-3 in Human Umbilical Vein Endothelial Cell Senescence

Whereas insulin-like growth factor binding protein-3 (IGFBP-3) is frequently upregulated in senescent replicatively exhausted human umbilical vein endothelial cells (HUVEC), a systematic analysis of four different HUVEC donors revealed that IGFBP-3 is not consistently upregulated in all isolates at senescence. Lentiviral overexpression of IGFBP-3 inhibited cell proliferation, induced apoptosis and senescence in young HUVEC. Knockdown of IGFBP-3 in senescent HUVEC by lentivirally expressed shRNA did not revert but rather enforced senescence-associated beta-galactosidase staining and apoptosis. Together the data suggest that, although IGFBP-3 acts as an anti-proliferative and premature senescence-inducing protein, the role of IGFBP-3 on senescence depends on the genetic background of the donor, and additional factors might be important to maintain the senescent phenotype.

Insulin-like growth factor binding protein-6 delays replicative senescence of human fibroblasts.

Highlights ► Proteomic analysis of senescent secretome reveals upregulation of IGFBP-6 in fibroblasts. ► IGFBP-6 knockdown induces premature senescence in young fibroblasts. ► IGFBP-6 lentiviral overexpression delays replicative senescence in fibroblasts.

Identification of Hsc70 as target for AGE modification in senescent human fibroblasts

Cellular senescence is known as a potent mechanism of tumor suppression, and cellular senescence in vitro also reflects at least some features of aging in vivo. The Free Radical Theory of aging suggests that reactive oxygen species are important causative agents of aging and cellular senescence. Besides damage of nucleic acids and lipids, also oxidative modifications of proteins have been described as potential causative events in the senescence response. However, the identity of protein targets for post-translational modifications in senescent cells has remained unclear. In the present communication, we analyzed the occurrence of oxidative posttranslational modifications in senescent human endothelial cells and dermal fibroblasts. We found a significant increase in the level of protein carbonyls and AGE modification with senescence in both cell types. Using 2D-Gel electrophoresis and Western Blot we found that heat shock cognate protein 70 is a bonafide target for AGE modification in human fibroblasts.

Two Functionally Distinct Isoforms of TL1A (TNFSF15) Generated by Differential Ectodomain Shedding

Tumor necrosis factor–like cytokine 1A (TL1A) is expressed in endothelial cells and contributes to T-cell activation, via an extracellular fragment TL1AL72-L251, generated by ectodomain shedding. Fragments of TL1A, referred to as vascular endothelial growth inhibitor, were found to induce growth arrest and apoptosis in endothelial cells; however, the underlying mechanisms remained obscure. Here, we show that full-length TL1A is the major detectable gene product in both human umbilical vein endothelial cells and circulating endothelial progenitor cells. TL1A expression was significantly enhanced in senescent circulating endothelial progenitor cells, and knockdown of TL1A partially reverted senescence. TL1A overexpression induced premature senescence in both circulating endothelial progenitor cells and human umbilical vein endothelial cells. We also identified a novel extracellular fragment of TL1A, TL1AV84-L251, resulting from differential ectodomain shedding, which induced growth arrest and apoptosis in human umbilical vein endothelial cells. These findings suggest that TL1A is involved in the regulation of endothelial cell senescence, via a novel fragment produced by differential ectodomain shedding.

Analysis of the cellular uptake and nuclear delivery of insulin-like growth factor binding protein-3 in human osteosarcoma cells.

Insulin‐like growth factor (IGF) binding protein‐3 (IGFBP‐3) regulates cell proliferation and survival by extracellular interaction and inactivation of the growth factor IGF‐I. Beyond that, IGF‐independent actions mediated by intracellular IGFBP‐3 including nuclear‐IGFBP‐3, have also been described. We here show, using both confocal and electron microscopy and cell fractionation, that the extracellular addition of IGFBP‐3 to living cells results in rapid uptake and nuclear delivery of IGFBP‐3, by yet partly unknown mechanisms. IGFBP‐3 is internalized through a dynamin‐dependent pathway, traffics through endocytic compartments and is finally delivered into the nucleus. We observed docking of IGFBP‐3 containing structures to the nuclear envelope and found IGFBP‐3 containing dot‐like structures to permeate the nuclear envelope. In summary, our findings establish the pathway by which this tumor suppressor protein is delivered from extracellular space to the nucleus.