Pidder Jansen-Dürr

p16INK4A is a robust in vivo biomarker of cellular aging in human skin

The cell‐cycle regulating gene, p16INK4A, encoding an inhibitor of cyclin‐dependent kinases 4 and 6, is considered to play an important role in cellular aging and in premature senescence. Although there is an age‐dependent increase of p16INK4A expression in human fibroblast senescence in vitro, no data are available regarding the age dependency of p16INK4A in vivo. To determine whether p16INK4A expression in human skin correlates with donor age, p16INK4A expression was analyzed by immunohistochemistry as well as the expression of the p16INK4A repressor BMI1. Samples from the age groups 0–20, 21–70, and 71–95 years were selected from a bank of healthy human skin. We show that the number of p16INK4A positive cells is significantly higher in elderly individuals compared to the younger age groups. The number of p16INK4A positive cells was found to be increased in both epidermis and dermis, compartments with strictly different proliferative activities. BMI1 gene expression was significantly down‐regulated with increasing donor age, whereas no striking age differences were observed for Ki67. In immunofluorescence co‐expression studies, Ki67‐positive cells were negative for p16INK4A and BMI1‐expressing cells also stained negatively for Ki67. In conclusion, we provide for the first time evidence that p16INK4A expression directly correlates with chronological aging of human skin in vivo. p16INK4A therefore is a biomarker for human aging in vivo. The data reported here suggest a model for changes in regulatory gene expression that drive aging in human skin.

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.

Replicative senescence of human endothelial cells in vitro involves G1 arrest, polyploidization and senescence-associated apoptosis

Human ageing is characterized by a progressive loss of physiological functions, increased tissue damage and defects in various tissue renewal systems. Age-related decreases of the cellular replicative capacity can be reproduced by in vitro assays of cellular ageing. When diploid human fibroblasts reach their finite lifespan, they enter an irreversible G1 growth arrest status referred to as replicative senescence. While deregulation of programmed cell death (apoptosis) is a key feature of age-related pathology in several tissues, this is not reflected in the standard in vitro senescence model of human fibroblasts, and the role of apoptosis during cellular ageing remains unclear. We have analyzed replicative senescence of human umbilical vein endothelial cells (HUVEC) in vitro and found that senescent HUVEC also arrest in the G1 phase of the cell cycle but, unlike fibroblasts, accumulate with a 4N DNA content, indicative of polyploidization. In contrast to human fibroblasts, senescent endothelial cells display a considerable increase in spontaneous apoptosis. The data imply that age-dependent apoptosis is a regular feature of human endothelial cells and suggest cell type specific differences in human ageing.

Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts

Limitation of lifespan in replicative senescence is related to oxidative stress, which is probably both the cause and consequence of impaired mitochondrial respiratory function. The respiration of senescent human diploid fibroblasts was analysed by high-resolution respirometry. To rule out cell-cycle effects, proliferating and growth-arrested young fibroblasts were used as controls. Uncoupled respiration, as normalized to citrate synthase activity, remained unchanged, reflecting a constant capacity of the respiratory chain. Oligomycin-inhibited respiration, however, was significantly increased in mitochondria of senescent cells, indicating a lower coupling of electron transport with phosphorylation. In contrast, growth-arrested young fibroblasts exhibited a higher coupling state compared with proliferating controls. In intact cells, partial uncoupling may lead to either decreased oxidative ATP production or a compensatory increase in routine respiration. To distinguish between these alternatives, we subtracted oligomycin-inhibited respiration from routine respiration, which allowed us to determine the part of respiratory activity coupled with ATP production. Despite substantial differences in the respiratory control ratio, ranging from 4 to 11 in the different experimental groups, a fixed proportion of respiratory capacity was maintained for coupled oxidative phosphorylation in all the experimental groups. This finding indicates that the senescent cells fully compensate for increased proton leakage by enhanced electron-transport activity in the routine state. These results provide a new insight into age-associated defects in mitochondrial function and compensatory mechanisms in intact cells.

ANCHORAGE-DEPENDENT TRANSCRIPTION OF THE CYCLIN A GENE

NIH 3T3 cells cultured in suspension fail to express cyclin A and hence cannot enter S phase and divide. We show that loss of cell adhesion to substratum abrogates cyclin A gene expression by blocking its promoter activity through the E2F site that mediates its cell cycle regulation in adherent cells. In suspended cells, G0-specific E2F complexes remain bound to the cyclin A promoter. Overexpression of cyclin D1 restores cyclin A transcription in suspended cells and rescues them from cell cycle arrest. In suspended cells, cyclin D1 and cyclin E accumulate normally upon serum stimulation, but their associated kinases remain inactive; their substrates, pRb and p107, are not hyperphosphorylated. Concomitantly, the cyclin-dependent kinase inhibitor, p27KIP1, is stabilized. Ectopic expression of p27KIP1 blocks cyclin A promoter activity through its EN binding site. These data suggest that the block to cyclin A transcription in nonadherent NIH 3T3 cells results from stabilization of p27KIP1 and subsequent inactivation of the specific E2F moiety required for its induction.

Metabolic analysis of senescent human fibroblasts reveals a role for AMP in cellular senescence.

Cellular senescence is considered a major tumour-suppressor mechanism in mammals, and many oncogenic insults, such as the activation of the ras proto-oncogene, trigger initiation of the senescence programme. Although it was shown that activation of the senescence programme involves the up-regulation of cell-cycle regulators such as the inhibitors of cyclin-dependent kinases p16INK4A and p21CIP-1, the mechanisms underlying the senescence response remain to be resolved. In the case of stress-induced premature senescence, reactive oxygen species are considered important intermediates contributing to the phenotype. Moreover, distinct alterations of the cellular carbohydrate metabolism are known to contribute to oncogenic transformation, as is best documented for the phenomenon of aerobic glycolysis. These findings suggest that metabolic alterations are involved in tumourigenesis and tumour suppression; however, little is known about the metabolic pathways that contribute to these processes. Using the human fibroblast model of in vitro senescence, we analysed age-dependent changes in the cellular carbohydrate metabolism. Here we show that senescent fibroblasts enter into a metabolic imbalance, associated with a strong reduction in the levels of ribonucleotide triphosphates, including ATP, which are required for nucleotide biosynthesis and hence proliferation. ATP depletion in senescent fibroblasts is due to dysregulation of glycolytic enzymes, and finally leads to a drastic increase in cellular AMP, which is shown here to induce premature senescence. These results suggest that metabolic regulation plays an important role during cellular senescence and hence tumour suppression.

p27KIP1 blocks cyclin E-dependent transactivation of cyclin A gene expression.

Cyclin E is necessary and rate limiting for the passage of mammalian cells through the G1 phase of the cell cycle. Control of cell cycle progression by cyclin E involves cdk2 kinase, which requires cyclin E for catalytic activity. Expression of cyclin E/cdk2 leads to an activation of cyclin A gene expression, as monitored by reporter gene constructs derived from the human cyclin A promoter. Promoter activation by cyclin E/cdk2 requires an E2F binding site in the cyclin A promoter. We show here that cyclin E/cdk2 kinase can directly bind to E2F/p107 complexes formed on the cyclin A promoter-derived E2F binding site, and this association is controlled by p27KIP1, most likely through direct protein-protein interaction. These observation suggest that cyclin E/cdk2 associates with E2F/p107 complexes in late G1 phase, once p27KIP1 has decreased below a critical threshold level. Since a kinase-negative mutant of cdk2 prevents promoter activation, it appears that transcriptional activation of the cyclin A gene requires an active cdk2 kinase tethered to its promoter region.

Senescence-associated cell death of human endothelial cells: the role of oxidative stress.

Replicative senescence of human endothelial cells was analyzed, using primary endothelial cells from the human umbilical vein endothelial cells (HUVEC) as an experimental model system. We had shown before that senescent HUVEC arrest in the G1 phase of the cell cycle and that a subpopulation of the senescent cells undergoes cell death. We now demonstrate that cell death occurs by apoptosis, characterized by activation of caspase 3. Using the redox-sensitive dye dihydrorhodamine 123, a significant accumulation of reactive oxygen species is detected in senescent but not young endothelial cells. To determine if increased oxidative stress may contribute to the senescent phenotype, cells were treated with tert-butyl hydroperoxide (tBHP), which is known to increase oxidative stress by decreasing the intracellular glutathione levels. We show here that mild tBHP stress induces a phenotype of premature senescence in a subpopulation of the treated cells, which closely resembles the phenotype of naturally senescent HUVEC, including growth arrest, senescence-associated beta-gal activity, and apoptotic cell death. These results establish a model of premature senescence for human endothelial cells, which will be suitable to analyze mechanisms of age-associated cell death.

Oxidative stress and mitochondrial impairment can be separated from lipofuscin accumulation in aged human skeletal muscle.

According to the free radical theory of aging, reactive oxygen species (ROS) act as a driving force of the aging process, and it is generally believed that mitochondrial dysfunction is a major source of increased oxidative stress in tissues with high content of mitochondria, such as muscle or brain. However, recent experiments in mouse models of premature aging have questioned the role of mitochondrial ROS production in premature aging. To address the role of mitochondrial impairment and ROS production for aging in human muscles, we have analyzed mitochondrial properties in muscle fibres isolated from the vastus lateralis of young and elderly donors. Mitochondrial respiratory functions were addressed by high‐resolution respirometry, and ROS production was analyzed by in situ staining with the redox‐sensitive dye dihydroethidium. We found that aged human skeletal muscles contain fully functional mitochondria and that the level of ROS production is higher in young compared to aged muscle. Accordingly, we could not find any increase in oxidative modification of proteins in muscle from elderly donors. However, the accumulation of lipofuscin was identified as a robust marker of human muscle aging. The data support a model, where ROS‐induced molecular damage is continuously removed, preventing the accumulation of dysfunctional mitochondria despite ongoing ROS production.

Effects of the human papilloma virus HPV-16 E7 oncoprotein on glycolysis and glutaminolysis: role of pyruvate kinase type M2 and the glycolytic-enzyme complex.

Proliferating and tumour cells express the glycolytic isoenzyme, pyruvate kinase type M2 (M2-PK), which occurs in a highly active tetrameric form and in a dimeric form with low affinity for phosphoenolpyruvate. The switch between the two forms regulates glycolytic phosphometabolite pools and the interaction between glycolysis and glutaminolysis. In the present study, we show the effects of oncoprotein E7 of the human papilloma virus (HPV)-16 (E7)-transformation on two NIH 3T3 cell strains with different metabolic characteristics. E7-transformation of the high glycolytic NIH 3T3 cell strain led to a shift of M2-PK to the dimeric form and, in consequence, to a decrease in the cellular pyruvate kinase mass-action ratio, the glycolytic flux rate and the (ATP+GTP)/(UTP+CTP) ratio, as well as to an increase in fructose 1,6-bisphosphate (FBP) levels, glutamine consumption and cell proliferation. The low glycolytic NIH 3T3 cell strain is characterized by high pyruvate and glutamine consumption rates and by an intrinsically large amount of the dimeric form of M2-PK, which is correlated with high FBP levels, a low (ATP+GTP)/(CTP+UTP) ratio and a high proliferation rate. E7-transformation of this cell strain led to an alteration in the glycolytic-enzyme complex that correlates with an increase in pyruvate and glutamine consumption and a slight increase in the flow of glucose to lactate. The association of phosphoglyceromutase within the glycolytic-enzyme complex led to an increase of glucose and serine consumption and a disruption of the linkage between glucose consumption and glutaminolysis. In both NIH 3T3 cell lines, transformation increased glutaminolysis and the positive correlation between alanine and lactate production.