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1.  Death by transposition – the enemy within? 
Here we present and develop the hypothesis that the derepression of endogenous retrotransposable elements (RTEs) – genomic parasites – is an important and hitherto under-unexplored molecular aging process that can potentially occur in most tissues. We further envision that the activation and continued presence of retrotransposition contribute to age-associated tissue degeneration and pathology. Chromatin is a complex and dynamic structure that needs to be maintained in a functional state throughout our lifetime. Studies of diverse species have revealed that chromatin undergoes extensive rearrangements during aging. Cellular senescence, an important component of mammalian aging, has recently been associated with decreased heterochromatinization of normally silenced regions of the genome. These changes lead to the expression of RTEs, culminating in their transposition. RTEs are common in all kingdoms of life, and comprise close to 50% of mammalian genomes. They are tightly controlled, as their activity is highly destabilizing and mutagenic to their resident genomes.
doi:10.1002/bies.201300097
PMCID: PMC3922893  PMID: 24129940
aging; anti-retroviral therapy; cellular senescence; retrotransposition
2.  Genomes of replicatively senescent cells undergo global epigenetic changes leading to gene silencing and activation of transposable elements 
Aging cell  2013;12(2):247-256.
Replicative cellular senescence is an important tumor suppression mechanism and also contributes to aging. Progression of both cancer and aging include significant epigenetic components, but the chromatin changes that take place during cellular senescence are not known. We used formaldehyde assisted isolation of regulatory elements (FAIRE) to map genome-wide chromatin conformations. In contrast to growing cells, whose genomes are rich with features of both open and closed chromatin, FAIRE profiles of senescent cells are significantly smoothened. This is due to FAIRE signal loss in promoters and enhancers of active genes, and FAIRE signal gain in heterochromatic gene poor regions. Chromatin of major retrotransposon classes, Alu, SVA and L1, becomes relatively more open in senescent cells, affecting most strongly the evolutionarily recent elements, and leads to an increase in their transcription and ultimately transposition. Constitutive heterochromatin in centromeric and peri-centromeric regions also becomes relatively more open, and the transcription of satellite sequences increases. The peripheral heterochromatic compartment (PHC) becomes less prominent, and centromere structure becomes notably enlarged. These epigenetic changes progress slowly after the onset of senescence, with some, such as mobilization of retrotransposable elements, becoming prominent only at late times. Many of these changes have also been noted in cancer cells.
doi:10.1111/acel.12047
PMCID: PMC3618682  PMID: 23360310
3.  Probing the depths of cellular senescence 
The Journal of Cell Biology  2013;202(1):11-13.
Cellular senescence is a state of irreversible cell cycle arrest that has been documented to both suppress cancer and promote aging. Although not well understood, extensive nuclear changes, including the remodeling of chromatin, take place as cells become senescent. In this issue, Ivanov et al. (2013. J. Cell Biol. http://dx.doi.org/jcb.201212110) report that chromatin fragments are released from the nuclei of senescent cells and are subsequently targeted for processing through the autophagy/lysosomal pathway.
doi:10.1083/jcb.201305155
PMCID: PMC3704986  PMID: 23816622
4.  Transposable elements become active and mobile in the genomes of aging mammalian somatic tissues 
Aging (Albany NY)  2013;5(12):867-883.
Transposable elements (TEs) were discovered by Barbara McClintock in maize and have since been found to be ubiquitous in all living organisms. Transposition is mutagenic and organisms have evolved mechanisms to repress the activity of their endogenous TEs. Transposition in somatic cells is very low, but recent evidence suggests that it may be derepressed in some cases, such as cancer development. We have found that during normal aging several families of retrotransposable elements (RTEs) start being transcribed in mouse tissues. In advanced age the expression culminates in active transposition. These processes are counteracted by calorie restriction (CR), an intervention that slows down aging. Retrotransposition is also activated in age-associated, naturally occurring cancers in the mouse. We suggest that somatic retrotransposition is a hitherto unappreciated aging process. Mobilization of RTEs is likely to be an important contributor to the progressive dysfunction of aging cells.
PMCID: PMC3883704  PMID: 24323947
Aging; epigenetics; chromatin; transposable elements
5.  The number of p16INK4a positive cells in human skin reflects biological age 
Aging cell  2012;11(4):722-725.
Summary
Cellular senescence is a defense mechanism in response to molecular damage which accumulates with aging. Correspondingly, the number of senescent cells has been reported to be greater in older than in younger subjects and furthermore associates with age-related pathologies. Inter-individual differences exist in the rate at which a person ages (biological age). Here, we studied whether younger biological age is related to fewer senescent cells in middle- aged individuals with the propensity for longevity, using p16INK4a as a marker for cellular senescence. We observed that a younger biological age associates with lower levels of p16INK4a positive cells in human skin.
doi:10.1111/j.1474-9726.2012.00837.x
PMCID: PMC3539756  PMID: 22612594
p16INK4a; biological age; familial longevity; cellular senescence
6.  How to measure RNA expression in rare senescent cells expressing any specific protein such as p16Ink4a 
Aging (Albany NY)  2013;5(2):120-129.
Here we describe a carefully optimized method for the preparation of high quality RNA by flow sorting of formaldehyde fixed senescent cells immunostained for any intracellular antigen. Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. The underlying cause of senescence due to replicative exhaustion is telomere shortening. We document here a spontaneous and apparently stochastic process that continuously generates senescent cells in cultures fully immortalized with telomerase. In the course of studying this phenomenon we developed a preparative fluorescence activated flow sorting method based on immunofluorescent staining of intracellular antigens that can also deliver RNA suitable for quantitative analysis of global gene expression. The protocols were developed using normal human diploid fibroblasts (HDF) and up to 5×107 cells could be conveniently processed in a single experiment. The methodology is based on formaldehyde crosslinking of cells, followed by permeabilization, antibody staining, flow sorting, reversal of the crosslinks, and recovery of the RNA. We explored key parameters such as crosslink reversal that affect the fragmentation of RNA. The recovered RNA is of high quality for downstream molecular applications based on short range sequence analysis, such qPCR, hybridization microarrays, and next generation sequencing. The RNA was analyzed by Affymetrix Gene Chip expression profiling and compared to RNA prepared by the direct lysis of cells. The correlation between the data sets was very high, indicating that the procedure does not introduce systematic changes in the mRNA transcriptome. The methods presented in this communication should be of interest to many investigators working in diverse model systems.
PMCID: PMC3616199  PMID: 23454889
Cellular senescence; flow cytometry; immunostaining; RNA purification; transcriptome profiling
7.  Accumulation of Senescent Cells in Mitotic Tissue of Aging Primates 
Cellular senescence, a stress induced growth arrest of somatic cells, was first documented in cell cultures over forty years ago, however its physiological significance has only recently been demonstrated. Using novel biomarkers of cellular senescence we examined whether senescent cells accumulate in tissues from baboons of ages encompassing the entire lifespan of this species. We show that dermal fibroblasts, displaying markers of senescence such as telomere damage, active checkpoint kinase ATM, high levels of heterochromatin proteins and elevated levels of p16, accumulate in skin biopsies from baboons with advancing age. The number of dermal fibroblasts containing damaged telomeres reaches a value of over 15% of total fibroblasts, whereas 80% of cells contain high levels of the heterochromatin protein HIRA. In skeletal muscle, a postmitotic tissue, only a small percentage of myonuclei containing damaged telomeres were detected regardless of animal age. The presence of senescent cells in mitotic tissues might therefore be a contributing factor to aging and age related pathology and provides further evidence that cellular senescence is a physiological event.
doi:10.1016/j.mad.2006.11.008
PMCID: PMC3654105  PMID: 17116315
Senescence; telomeres; DNA damage; aging; p16; p21
8.  Why do we grow old: is it because our cells just wear out, we run out of cells (or both), and what can we do about it? 
Longevity & Healthspan, a new BioMed Central journal, has launched a thematic series on cellular senescence and aging, a quickly evolving field critical to our understanding of the biology of aging.
doi:10.1186/2046-2395-2-7
PMCID: PMC3922936  PMID: 24472649
9.  Kinetic profiling of the c-Myc transcriptome and bioinformatic analysis of repressed gene promoters 
Cell Cycle  2011;10(13):2184-2196.
Mammalian c-Myc is a member of a small family of three related proto-oncogenic transcription factors. c-Myc has an unusually broad array of regulatory functions, which include roles in cell cycle and apoptosis, a variety of metabolic functions, cell differentiation, senescence and stem cell maintenance. c-Myc modulates the expression of a very large number of genes, but the magnitude of the majority of the regulatory effects is only two-fold or less. c-Myc can both activate and repress the promoters of its target genes. Identification of genes directly regulated by c-Myc has been an enduring question in the field. We report here microarray expression profiling of a high resolution time course of c-Myc induction, using fibroblast cells in which c-Myc activity can be modulated from null to physiological. The c-Myc transcriptome data set presented is the largest reported to date with 4,186 differentially regulated genes (1,826 upregulated, 2,360 downregulated, 1% FDR). The gene expression patterns fit well with the known biological functions of c-Myc. We describe several novel findings and present tools for further data mining. Although the mechanisms of transcriptional activation by c-Myc are well understood, how c-Myc represses an even greater number of genes remains incompletely described. One mechanism involves the binding of c-Myc to other, positively acting transcription factors and interfering with their activities. We identified rapid-response genes likely to be direct c-Myc targets and analyzed the promoters of the repressed genes to identify transcription factors that could be targets of c-Myc repression.
doi:10.4161/cc.10.13.16249
PMCID: PMC3154366  PMID: 21623162
c-Myc; proto-oncogene; transcriptome; expression profiling; microarray; promoter; gene regulation
10.  Age-associated increase in heterochromatic marks in murine and primate tissues 
Aging cell  2010;10(2):292-304.
Summary
Chromatin is highly dynamic and subject to extensive remodeling under many physiological conditions. Changes in chromatin that occur during the aging process are poorly documented and understood in higher organisms, such as mammals. We developed an immunofluorescence assay to quantitatively detect, at the single cell level, changes in the nuclear content of chromatin-associated proteins. We find increased levels of the heterochromatin-associated proteins histone macro H2A (mH2A) and heterochromatin protein 1 beta (HP1β) in human fibroblasts during replicative senescence in culture, and for the first time, an age-associated increase in these heterochromatin marks in several tissues of mice and primates. Mouse lung was characterized by monophasic mH2A expression histograms at both ages, and an increase in mean staining intensity at old age. In the mouse liver we observed increased age-associated localization of mH2A to regions of pericentromeric heterochromatin. In skeletal muscle we found two populations of cells with either low or high mH2A levels. This pattern of expression was similar in mouse and baboon, and showed a clear increase in the proportion of nuclei with high mH2A levels in older animals. The frequencies of cells displaying evidence of increased heterochromatinization are too high to be readily accounted for by replicative or oncogene-induced cellular senescence, and are prominently found in terminally differentiated, post mitotic tissues that are not conventionally thought to be susceptible to senescence. Our findings distinguish specific chromatin states in individual cells of mammalian tissues, and provide a foundation to further investigate the progressive epigenetic changes that occur during aging.
doi:10.1111/j.1474-9726.2010.00666.x
PMCID: PMC3079313  PMID: 21176091
11.  A link between the accumulation of DNA damage and loss of multi-potency of human mesenchymal stromal cells 
Human mesenchymal stromal cells (hMSCs) represent an attractive cell source for clinic applications. Besides being multi-potent, recent clinical trials suggest that they secrete both trophic and immunomodulatory factors, allowing allogenic MSCs to be used in a wider variety of clinical situations. The yield of prospective isolation is however very low, making expansion a required step toward clinical applications. Unfortunately, this leads to a significant decrease in their stemness. To identify the mechanism behind loss of multi-potency, hMSCs were expanded until replicative senescence and the concomitant molecular changes were characterized at regular intervals. We observed that, with time of culture, loss of multi-potency was associated with both the accumulation of DNA damage and the respective activation of the DNA damage response pathway, suggesting a correlation between both phenomena. Indeed, exposing hMSCs to DNA damage agents led to a significant decrease in the differentiation potential. We also showed that hMSCs are susceptible to accumulate DNA damage upon in vitro expansion, and that although hMSCs maintained an effective nucleotide excision repair activity, there was a progressive accumulation of DNA damage. We propose a model in which DNA damage accumulation contributes to the loss of differentiation potential of hMSCs, which might not only compromise their potential for clinical applications but also contribute to the characteristics of tissue ageing.
doi:10.1111/j.1582-4934.2009.00931.x
PMCID: PMC3539748  PMID: 19818093
ageing; senescence; human mesenchymal stem cells; DNA damage; in vitro expansion; clinical application
12.  Phosphatidylenthanolamine Binding Protein aka Raf Kinase Inhibitor Protein: A Brief History of Its Discovery and the Remarkable Diversity of Biological Functions 
Phosphatidylethanolamine-binding protein (PEBP) was identified almost three decades ago as an abundant protein in bovine brain. PEBP is the prototype of a highly conserved family of proteins represented in all three major phylogenetic divisions, eukaryota, bacteria, and archaea, with no significant sequence homology to other proteins. PEBP proteins have been studied in many species. The most thoroughly explored biological role of PEBP is that of a modulator of intracellular signaling pathways, which is mediated by its ability to bind and inhibit a number of protein kinases. The first such interaction that came to light was with the Raf1 kinase, and PEBP is thus widely referred to in the literature under its alternate name RKIP (Raf kinase inhibitory protein). The activity of RKIP itself is subject to regulation by phosphorylation. Intriguingly, PEBP has also been reported to possess additional, and diverse, biological functions unrelated to protein kinase networks that remain to be investigated in detail. Recent findings that RKIP may function as a suppressor of cancer metastasis are of great interest and importance. Prognostic and therapeutic applications of RKIP in human cancer were the subject of the first international workshop on RKIP that was held at the University of California, Los Angeles, in March 2010. This paper was presented at the workshop as a summary of the history of this still small but rapidly evolving field.
doi:10.1615/ForumImmunDisTher.v2.i1.20
PMCID: PMC3516847  PMID: 23227430
cancer; signal transduction; metastasis; tumor suppression; phosphorylation; apoptosis; MAP kinase pathway; NF-κB pathway; G protein–coupled receptor pathway
13.  Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay 
Aging (Albany NY)  2011;3(10):955-967.
Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.
PMCID: PMC3229972  PMID: 22006542
Aging; cellular senescence; quantitative protein assay; fluorescence microscopy; NanoOrange® reagent
14.  Proteomic profiling of Myc-associated proteins 
Cell cycle (Georgetown, Tex.)  2010;9(24):4908-4921.
Mammalian c-Myc is a member of a small family of three closely related transcription factors. The Myc family of protooncogenes are among the most potent activators of tumorigenesis and are frequently overexpressed in diverse cancers. c-Myc has an unusually broad array of regulatory functions, which include, in addition to roles in the cell cycle and apoptosis, effects on a variety of metabolic functions, cell differentiation, senescence and stem cell maintenance. A significant number of c-Myc interacting proteins have already been defined, but it is widely believed that the c-Myc interactome is vastly larger than currently documented. In addition to interactions with components of the transcription machinery, transcription independent nuclear interactions with the DNA replication and RNA processing pathways have been reported. Cytoplasmic roles of c-Myc have also been recently substantiated. Recent advances in proteomics have opened new possibilities for the isolation of protein complexes under native conditions and confidently identifying the components using ultrasensitive, high mass accuracy and high resolution mass spectrometry techniques. In this communication we report a new tandem affinity purification (TAP) c-Myc interaction screen that employed new cell lines with near-physiological levels of c-Myc expression with multi-dimensional protein identification techniques (MudPIT) for the detection and quantification of proteins. Both label-free and the recently developed stable isotope labeling with amino acids in cell culture (SILAC) methodologies were used. Combined data from multiple biological replicates provided a dataset of 418 non-redundant proteins, 389 of which are putative novel interactors. This new information should significantly advance our understanding of this interesting and important master regulator.
PMCID: PMC3047814  PMID: 21150319
c-Myc; proto-oncogene; proteomics; tandem affinity purification; MudPIT analysis; SILAC; protein-protein interaction; protein complex
15.  Proteomic profiling of Myc-associated proteins 
Cell Cycle  2010;9(24):4908-4921.
Mammalian c-Myc is a member of a small family of three closely related transcription factors. The Myc family of proto-oncogenes are among the most potent activators of tumorigenesis and are frequently overexpressed in diverse cancers. c-Myc has an unusually broad array of regulatory functions, which include, in addition to roles in the cell cycle and apoptosis, effects on a variety of metabolic functions, cell differentiation, senescence and stem cell maintenance. A significant number of c-Myc interacting proteins have already been defined, but it is widely believed that the c-Myc interactome is vastly larger than currently documented. In addition to interactions with components of the transcription machinery, transcription independent nuclear interactions with the DNA replication and RNA processing pathways have been reported. Cytoplasmic roles of c-Myc have also been recently substantiated. Recent advances in proteomics have opened new possibilities for the isolation of protein complexes under native conditions and confidently identifying the components using ultrasensitive, high mass accuracy and high resolution mass spectrometry techniques. In this communication we report a new tandem affinity purification (TAP) c-Myc interaction screen that employed new cell lines with near-physiological levels of c-Myc expression with multi-dimensional protein identification techniques (MudPIT) for the detection and quantification of proteins. Both label-free and the recently developed stable isotope labeling with amino acids in cell culture (SILAC) methodologies were used. Combined data from multiple biological replicates provided a dataset of 418 non-redundant proteins, 389 of which are putative novel interactors. This new information should significantly advance our understanding of this interesting and important master regulator.
doi:10.4161/cc.9.24.14199
PMCID: PMC3047814  PMID: 21150319
c-Myc; proto-oncogene; proteomics; tandem affinity purification; MudPIT analysis; SILAC; protein-protein interaction; protein complex
16.  Regulation of RKIP Function by Helicobacter pylori in Gastric Cancer 
PLoS ONE  2012;7(5):e37819.
Helicobacter pylori (H. pylori) is a gram-negative, spiral-shaped bacterium that infects more than half of the world’s population and is a major cause of gastric adenocarcinoma. The mechanisms that link H. pylori infection to gastric carcinogenesis are not well understood. In the present study, we report that the Raf-kinase inhibitor protein (RKIP) has a role in the induction of apoptosis by H. pylori in gastric epithelial cells. Western blot and luciferase transcription reporter assays demonstrate that the pathogenicity island of H. pylori rapidly phosphorylates RKIP, which then localizes to the nucleus where it activates its own transcription and induces apoptosis. Forced overexpression of RKIP enhances apoptosis in H. pylori-infected cells, whereas RKIP RNA inhibition suppresses the induction of apoptosis by H. pylori infection. While inducing the phosphorylation of RKIP, H. pylori simultaneously targets non-phosphorylated RKIP for proteasome-mediated degradation. The increase in RKIP transcription and phosphorylation is abrogated by mutating RKIP serine 153 to valine, demonstrating that regulation of RKIP activity by H. pylori is dependent upon RKIP’s S153 residue. In addition, H. pylori infection increases the expression of Snail, a transcriptional repressor of RKIP. Our results suggest that H. pylori utilizes a tumor suppressor protein, RKIP, to promote apoptosis in gastric cancer cells.
doi:10.1371/journal.pone.0037819
PMCID: PMC3360604  PMID: 22662230
17.  Cellular senescence and organismal aging 
Mechanisms of Ageing and Development  2008;129(7-8):467-474.
Cellular senescence, first observed and defined using in vitro cell culture studies, is an irreversible cell cycle arrest which can be triggered by a variety of factors. Emerging evidence suggests that cellular senescence acts as an in vivo tumor suppression mechanism by limiting aberrant proliferation. It has also been postulated that cellular senescence can occur independently of cancer and contribute to the physiological processes of normal organismal aging. Recent data have demonstrated the in vivo accumulation of senescent cells with advancing age. Some characteristics of senescent cells, such as the ability to modify their extracellular environment, could play a role in aging and age related pathology. In this review, we examine current evidence that links cellular senescence and organismal aging.
doi:10.1016/j.mad.2008.04.001
PMCID: PMC3297662  PMID: 18502472
Cellular senescence; Aging; Telomeres
18.  Postnatal liver growth and regeneration are independent of c-myc in a mouse model of conditional hepatic c-myc deletion 
BMC Physiology  2012;12:1.
Background
The transcription factor c-myc regulates genes involved in hepatocyte growth, proliferation, metabolism, and differentiation. It has also been assigned roles in liver development and regeneration. In previous studies, we made the unexpected observation that c-Myc protein levels were similar in proliferating fetal liver and quiescent adult liver with c-Myc displaying nucleolar localization in the latter. In order to investigate the functional role of c-Myc in adult liver, we have developed a hepatocyte-specific c-myc knockout mouse, c-mycfl/fl;Alb-Cre.
Results
Liver weight to body weight ratios were similar in control and c-myc deficient mice. Liver architecture was unaffected. Conditional c-myc deletion did not result in compensatory induction of other myc family members or in c-Myc's binding partner Max. Floxed c-myc did have a negative effect on Alb-Cre expression at 4 weeks of age. To explore this relationship further, we used the Rosa26 reporter line to assay Cre activity in the c-myc floxed mice. No significant difference in Alb-Cre activity was found between control and c-mycfl/fl mice. c-myc deficient mice were studied in a nonproliferative model of liver growth, fasting for 48 hr followed by a 24 hr refeeding period. Fasting resulted in a decrease in liver mass and liver protein, both of which recovered upon 24 h of refeeding in the c-mycfl/fl;Alb-Cre animals. There was also no effect of reducing c-myc on recovery of liver mass following 2/3 partial hepatectomy.
Conclusions
c-Myc appears to be dispensable for normal liver growth during the postnatal period, restoration of liver mass following partial hepatectomy and recovery from fasting.
doi:10.1186/1472-6793-12-1
PMCID: PMC3353165  PMID: 22397685
19.  Epigenetic Control of Aging 
Antioxidants & Redox Signaling  2011;14(2):241-259.
Abstract
Organismal aging and longevity are influenced by many complex interacting factors. Epigenetics has recently emerged as another possible determinant of aging. Here, we review some of the epigenetic pathways that contribute to cellular senescence and age-associated phenotypes. Strategies aimed to reverse age-linked epigenetic alterations may lead to the development of new therapeutic interventions to delay or alleviate some of the most debilitating age-associated diseases. Antioxid. Redox Signal. 14, 241–259.
doi:10.1089/ars.2010.3250
PMCID: PMC3014766  PMID: 20518699
20.  Analysis of Cell Cycle Phases and Progression in Cultured Mammalian Cells 
Methods (San Diego, Calif.)  2007;41(2):143-150.
Fluorescence Activated Cell Sorting (FACS) analysis has become a standard tool to analyze cell cycle distributions in populations of cells. These methods require relatively large numbers of cells, and do not provide optimal resolution of the transitions between cell cycle phases. In this report we describe in detail complementary methods that utilize the incorporation of nucleotide analogs combined with microscopic examination. While often more time consuming, these protocols typically require far fewer cells, and allow accurate kinetic assessment of cell cycle progression. We also describe the use of a technique for the synchronization of adherent cells in mitosis by simple mechanical agitation (mitotic shake-off) that eliminates physiological perturbation associated with drug treatments.
doi:10.1016/j.ymeth.2006.07.022
PMCID: PMC1828876  PMID: 17189856
21.  Widespread expression of the Supv3L1 mitochondrial RNA helicase in the mouse 
Transgenic research  2009;19(4):691-701.
Supv3L1 is an evolutionarily conserved helicase that plays a critical role in the mitochondrial RNA surveillance and degradation machinery. Conditional ablation of Supv3L1 in adult mice leads to premature aging phenotypes including loss of muscle mass and adipose tissue and severe skin abnormalities. To get insights into the spatial and temporal expression of Supv3L1 in the mouse, we generated knock-in and transgenic strains in which an EGFP reporter was placed under control of the Supv3L1 native promoter. During development, expression of Supv3L1 begins at the blastocyst stage, becomes widespread and strong in all fetal tissues and cell types, and continues during postnatal growth. In mature animals reporter expression is only slightly diminished in most tissues and continues to be highly expressed in the brain, peripheral sensory organs, and testis. Together, these data confirm that Supv3L1 is an important developmentally regulated gene, which continues to be expressed in all mature tissues, particularly the rapidly proliferating cells of testes, but also in the brain and sensory organs. The transgenic mice and cell lines derived from them constitute a valuable tool for the examination of the spatial and temporal aspects of Supv3L1 promoter activity, and should facilitate future screens for small molecules that regulate Supv3L1 expression.
doi:10.1007/s11248-009-9346-0
PMCID: PMC2888911  PMID: 19937380
Supv3L1; Suv3; mouse; expression pattern; retina; EGFP reporter
22.  Disruption of Supv3L1 damages the skin, and causes sarcopenia, loss of fat, and death 
Supv3L1 is a conserved and ubiquitously expressed helicase found in numerous tissues and cell types of many species. In human cells, SUPV3L1 was shown to suppress apoptotic death, sister chromatid exchange, and impair mitochondrial RNA metabolism and protein synthesis. In vitro experiments revealed binding of SUPV3L1 to BLM and WRN proteins suggesting a role in genome maintenance processes. Disruption of the Supv3L1 gene in the mouse has been reported to be embryonic lethal at early developmental stages. We generated a conditional mouse in which the phenotypes associated with the removal of exon 14 can be tested in a variety of tissues. Disruption mediated by a Mx1 promoter-driven Cre displayed a postnatal growth delay, reduced life span, loss of adipose tissue, muscle mass, and severe skin abnormalities manifesting as ichthyosis, thickening of the epidermis, and atrophy of the dermis and subcutaneous tissue. Using a tamoxifen-activatable Esr1/Cre driver, Supv3L1 disruption resulted in growth retardation and aging phenotypes including loss of adipose tissue, muscle mass, kyphosis, cachexia and premature death. Many of the abnormalities seen in the Mx1-Cre mice, such as hyperkeratosis characterized by profound scaling of feet and tail, could also be detected in tamoxifen-inducible Cre mice. Conditional ablation of Supv3L1 in keratinocytes confirmed atrophic changes in the skin and ichthyosis-like changes. Together, these data indicate that Supv3L1 is important for the maintenance of the skin barrier. Additionally, loss of Supv3L1 function leads to accelerated aging-like phenotypes.
doi:10.1007/s00335-008-9168-z
PMCID: PMC2667228  PMID: 19145458
23.  Mechanism of Suppression of the Raf/MEK/Extracellular Signal-Regulated Kinase Pathway by the Raf Kinase Inhibitor Protein 
Molecular and Cellular Biology  2000;20(9):3079-3085.
We have recently identified the Raf kinase inhibitor protein (RKIP) as a physiological endogenous inhibitor of the Raf-1/MEK/extracellular signal-regulated kinase (ERK) pathway. RKIP interfered with MEK phosphorylation and activation by Raf-1, resulting in the suppression of both Raf-1-induced transformation and AP-1-dependent transcription. Here we report the molecular mechanism of RKIP's inhibitory function. RKIP can form ternary complexes with Raf-1, MEK, and ERK. However, whereas MEK and ERK can simultaneously associate with RKIP, Raf-1 binding to RKIP and that of MEK are mutually exclusive. RKIP is able to dissociate a Raf-1–MEK complex and behaves as a competitive inhibitor of MEK phosphorylation. Mapping of the binding domains showed that MEK and Raf-1 bind to overlapping sites in RKIP, whereas MEK and RKIP associate with different domains in Raf-1, and Raf-1 and RKIP bind to different sites in MEK. Both the Raf-1 and the MEK binding sites in RKIP need to be destroyed in order to relieve RKIP-mediated suppression of the Raf-1/MEK/ERK pathway, indicating that binding of either Raf-1 or MEK is sufficient for inhibition. The properties of RKIP reveal the specific sequestration of interacting components as a novel motif in the cell's repertoire for the regulation of signaling pathways.
PMCID: PMC85596  PMID: 10757792
24.  Aging by epigenetics - a consequence of chromatin damage? 
Experimental cell research  2008;314(9):1909-1917.
Chromatin structure is not fixed. Instead, chromatin is dynamic and is subject to extensive developmental and age-associated remodeling. In some cases, this remodeling appears to counter the aging and age-associated diseases, such as cancer, and extend organismal lifespan. However, stochastic non-deterministic changes in chromatin structure might, over time, also contribute to the break down of nuclear, cell and tissue function, and consequently aging and age-associated diseases.
doi:10.1016/j.yexcr.2008.02.023
PMCID: PMC2464300  PMID: 18423606
25.  Signaling Crossroads: The function of Raf Kinase Inhibitory Protein in Cancer, the Central Nervous System and Reproduction 
Cellular signalling  2007;20(1):1-9.
The Raf kinase inhibitory protein 1 (RKIP-1) and its orthologs are conserved throughout evolution and widely expressed in eukaryotic organisms. In its non-phosphorylated form RKIP-1 negatively regulates the Raf/MEK/ERK pathway by interfering with the activity of Raf-1. In its phosphorylated state, RKIP-1 dissociates from Raf-1 and inhibits GRK-2, a negative regulator of G-protein coupled receptors (GPCRs). Available data indicate that the phosphorylation of RKIP-1 by PKC can stimulate both the Raf/MEK/ERK and GPCR pathways. RKIP-1 has also been implicated as a negative regulator of the NF-κB pathway. Recent studies have shown that phosphorylated RKIP-1 binds to the centrosomal and kinetochore regions of metaphase chromosomes, where it may be involved in regulating the partitioning of chromosomes and the progression through mitosis. The collective evidence indicates that RKIP-1 regulates the activity and mediates the crosstalk between several important cellular signaling pathways. A variety of ablative interventions suggest that reduced RKIP-1 function may influence metastasis, angiogenesis, resistance to apoptosis, and genome integrity. Attenuation of RKIP-1 may also affect cardiac and neurological functions, spermatogenesis, sperm decapactiation, and reproductive behavior. In this review, the role of RKIP-1 in cellular signaling, and especially its functions revealed using a mouse knockout model, are discussed.
doi:10.1016/j.cellsig.2007.07.003
PMCID: PMC2231335  PMID: 17706925
RKIP-1; PEBP; signaling; mouse model; olfaction; reproduction; metastases

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