CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system-New mode of action for caffeine.

We show that the cyclin-dependent kinase inhibitor 1B (CDKN1B)/p27, previously known as a cell cycle inhibitor, is also localized within mitochondria. The migratory capacity of endothelial cells, which need intact mitochondria, is completely dependent on mitochondrial p27. Mitochondrial p27 improves mitochondrial membrane potential, increases adenosine triphosphate (ATP) content, and is required for the promigratory effect of caffeine. Domain mapping of p27 revealed that the N-terminus and C-terminus are required for those improvements. Further analysis of those regions revealed that the translocation of p27 into the mitochondria and its promigratory activity depend on serine 10 and threonine 187. In addition, mitochondrial p27 protects cardiomyocytes against apoptosis. Moreover, mitochondrial p27 is necessary and sufficient for cardiac myofibroblast differentiation. In addition, p27 deficiency and aging decrease respiration in heart mitochondria. Caffeine does not increase respiration in p27-deficient animals, whereas aged mice display improvement after 10 days of caffeine in drinking water. Moreover, caffeine induces transcriptome changes in a p27-dependent manner, affecting mostly genes relevant for mitochondrial processes. Caffeine also reduces infarct size after myocardial infarction in prediabetic mice and increases mitochondrial p27. Our data characterize mitochondrial p27 as a common denominator that improves mitochondria-dependent processes and define an increase in mitochondrial p27 as a new mode of action of caffeine.

Impingement Syndrome of the Shoulder.

BACKGROUND: Shoulder pain is the third most common musculoskeletal complaint in orthopedic practice. It is usually due to a defect of the rotator cuff and/or an impingement syndrome. METHODS: This review is based on pertinent literature retrieved by a selective search of the Medline database. RESULTS: Patients with shoulder impingement syndrome suffer from painful entrapment of soft tissue whenever they elevate the arm. The pathological mechanism is a structural narrowing in the subacromial space. A multiplicity of potential etiologies makes the diagnosis more difficult; it is established by the history and physical examination and can be confirmed with x-ray, ultra - sonography, and magnetic resonance imaging. The initial treatment is conservative, e.g., with nonsteroidal antiinflammatory drugs, infiltrations, and patient exercises. Conservative treatment yields satisfactory results within 2 years in 60% of cases. If symptoms persist, decompressive surgery is performed as long as the continuity of the rotator cuff is preserved and there is a pathological abnormality of the bursa. The correct etiologic diagnosis and choice of treatment are essential for a good outcome. The formal evidence level regarding the best treatment strategy is low, and it has not yet been determined whether surgical or conservative treatment is better. CONCLUSION: Randomized controlled therapeutic trials are needed so that a standardized treatment regimen can be established.

The Anti-Apoptotic Properties of APEX1 in the Endothelium Require the First 20 Amino Acids and Converge on Thioredoxin-1.

The APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1) has a disordered N-terminus, a redox, and a DNA repair domain. APEX1 has anti-apoptotic properties, which have been linked to both domains depending on cell type and experimental conditions. AIMS: As protection against apoptosis is a hallmark of vessel integrity, we wanted to elucidate whether APEX1 acts anti-apoptotic in primary human endothelial cells and, if so, what the underlying mechanisms are. RESULTS: APEX1 inhibits apoptosis in endothelial cells by reducing Cathepsin D (CatD) cleavage, potentially by binding to the unprocessed form. Diminished CatD activation results in increased Thioredoxin-1 protein levels leading to reduced Caspase 3 activation. Consequently, apoptosis rates are decreased. This depends on the first twenty amino acids in APEX1, because APEX1 (21-318) induces CatD activity, decreases Thioredoxin-1 protein levels, and, thus, increases Caspase 3 activity and apoptosis. Along the same lines, APEX1 (1-20) inhibits Caspase 3 cleavage and apoptosis. Furthermore, re-expression of Thioredoxin-1 via lentiviral transduction rescues endothelial cells from APEX1 (21-318)-induced apoptosis. In an in vivo model of restenosis, which is characterized by oxidative stress, endothelial activation, and smooth muscle cell proliferation, Thioredoxin-1 protein levels are reduced in the endothelium of the carotids. INNOVATION: APEX1 acts anti-apoptotic in endothelial cells. This anti-apoptotic effect depends on the first 20 amino acids of APEX1. CONCLUSION: As proper function of the endothelium during life span is a hallmark for individual health span, a detailed characterization of the functions of the APEX1N-terminus is required to understand all its cellular properties. Antioxid. Redox Signal. 26, 616-629.

MicroRNA-15b regulates mitochondrial ROS production and the senescence-associated secretory phenotype through sirtuin 4/SIRT4.

Mammalian sirtuins are involved in the control of metabolism and life-span regulation. Here, we link the mitochondrial sirtuin SIRT4 with cellular senescence, skin aging, and mitochondrial dysfunction. SIRT4 expression significantly increased in human dermal fibroblasts undergoing replicative or stress-induced senescence triggered by UVB or gamma-irradiation. In-vivo, SIRT4 mRNA levels were upregulated in photoaged vs. non-photoaged human skin. Interestingly, in all models of cellular senescence and in photoaged skin, upregulation of SIRT4 expression was associated with decreased levels of miR-15b. The latter was causally linked to increased SIRT4 expression because miR-15b targets a functional binding site in the SIRT4 gene and transfection of oligonucleotides mimicking miR-15b function prevented SIRT4 upregulation in senescent cells. Importantly, increased SIRT4 negatively impacted on mitochondrial functions and contributed to the development of a senescent phenotype. Accordingly, we observed that inhibition of miR-15b, in a SIRT4-dependent manner, increased generation of mitochondrial reactive oxygen species, decreased mitochondrial membrane potential, and modulated mRNA levels of nuclear encoded mitochondrial genes and components of the senescence-associated secretory phenotype (SASP). Thus, miR-15b is a negative regulator of stress-induced SIRT4 expression thereby counteracting senescence associated mitochondrial dysfunction and regulating the SASP and possibly organ aging, such as photoaging of human skin.

The aryl hydrocarbon receptor promotes aging phenotypes across species.

The ubiquitously expressed aryl hydrocarbon receptor (AhR) induces drug metabolizing enzymes as well as regulators of cell growth, differentiation and apoptosis. Certain AhR ligands promote atherosclerosis, an age-associated vascular disease. Therefore, we investigated the role of AhR in vascular functionality and aging. We report a lower pulse wave velocity in young and old AhR-deficient mice, indicative of enhanced vessel elasticity. Moreover, endothelial nitric oxide synthase (eNOS) showed increased activity in the aortas of these animals, which was reflected in increased NO production. Ex vivo, AhR activation reduced the migratory capacity of primary human endothelial cells. AhR overexpression as well as treatment with a receptor ligand, impaired eNOS activation and reduced S-NO content. All three are signs of endothelial dysfunction. Furthermore, AhR expression in blood cells of healthy human volunteers positively correlated with vessel stiffness. In the aging model Caenorhabditis elegans, AhR-deficiency resulted in increased mean life span, motility, pharynx pumping and heat shock resistance, suggesting healthier aging. Thus, AhR seems to have a negative impact on vascular and organismal aging. Finally, our data from human subjects suggest that AhR expression levels could serve as an additional, new predictor of vessel aging.

Cellular functions of the dual-targeted catalytic subunit of telomerase, telomerase reverse transcriptase--potential role in senescence and aging.

Over the last 40 years it has become clear that telomeres, the end of the chromosomes, and the enzyme telomerase reverse transcriptase (TERT), which is required to counteract their shortening, play a pivotal role in senescence and aging. However, over the last years several studies demonstrated that TERT belongs to the group of dual-targeted proteins. It contains a bipartite nuclear localization signal as well as a mitochondrial targeting sequence and, under physiological conditions, is found in both organelles in several cell types including terminally differentiated, post-mitotic cells. The canonical function of TERT is to prevent telomere erosion and thereby the development of replicative senescence and genetic instability. Besides telomere extension, TERT exhibits other non-telomeric activities such as cell cycle regulation, modulation of cellular signaling and gene expression, augmentation of proliferative lifespan as well as DNA damage responses. Mitochondrial TERT is able to reduce reactive oxygen species, mitochondrial DNA damage and apoptosis. Because of the localization of TERT in the nucleus and in the mitochondria, it must have different functions in the two organelles as mitochondrial DNA does not contain telomeric structures. However, the organelle-specific functions are not completely understood. Strikingly, the regulation by phosphorylation of TERT seems to reveal multiple parallels. This review will summarize the current knowledge about the cellular functions and post-translational regulation of the dual-targeted protein TERT.

The imbalanced redox status in senescent endothelial cells is due to dysregulated Thioredoxin-1 and NADPH oxidase 4.

Environmental stressors as well as genetic modifications are known to enhance oxidative stress and aging processes. Mitochondrial and nuclear dysfunctions contribute to the onset of aging. One of the most important redox regulators in primary human endothelial cells is Thioredoxin-1 (Trx-1), a 12 kD protein with additional anti-apoptotic properties. Cellular generators of reactive oxygen species are NADPH oxidases (NOXs), of which NOX4 shows highest expression levels in endothelial cells. Therefore, the aim of the study was to investigate how Trx-1 and NOX4 are regulated during stress-induced premature senescence in endothelial cells. We treated primary human endothelial cells for two weeks with H2O2 to generate stress-induced premature senescence in these cells. In this model senescence-associated ß-Galactosidase and nuclear p21 as senescence markers are increased. Moreover, total and mitochondrial reactive oxygen species formation is enhanced. An imbalanced redox homeostasis is detected by elevated NOX4 and decreased Trx-1 levels. This can be rescued by lentiviral expression of Trx-1. Moreover, the lysosomal protease Cathepsin D is over-activated, which results in reduced Trx-1 protein levels. Inhibition of "over-active" Cathepsin D by the specific, cell-permeable inhibitor pepstatin A abolishes the increase in nuclear p21 protein, ROS formation and degradation of Trx-1 protein, thus leading to blockade of stress-induced premature senescence by stabilizing the cellular redox homeostasis. Aortic Trx-1 levels are decreased and Cathepsin D activity is increased in NOX4 transgenic mice exclusively expressing NOX4 in the endothelium when compared to their wildtype littermates. Thus, loss of Trx-1 and upregulation of NOX4 importantly contribute to the imbalance in the redox-status of senescent endothelial cells ex vivo and in vivo.

The cytokine midkine supports neutrophil trafficking during acute inflammation by promoting adhesion via ß2 integrins (CD11/CD18).

Emerging evidence suggests a role of the cytokine midkine (MK) in inflammation. In this study, its functional relevance for recruitment of polymorphonuclear neutrophils (PMNs) during acute inflammation was investigated. Intravital microscopy and histologic analysis of tumor necrosis factor-α-stimulated cremaster muscle venules revealed severely compromised leukocyte adhesion and extravasation in MK(-/-) mice compared with MK(+/+) animals. Systemic administration of recombinant MK completely rescued the adhesion defect in MK(-/-) mice. In a hind limb ischemia model, leukocyte accumulation in MK(-/-) mice was significantly diminished compared with MK(+/+) animals. However, MK did not lead to an inflammatory activation of PMNs or endothelial cells suggesting that it does not serve as classical proinflammatory cytokine. Unexpectedly, immobilized MK mediated PMN adhesion under static and flow conditions, whereas PMN-derived MK was dispensable for the induction of adhesion. Furthermore, adhesion strengthening remained unaffected by MK. Flow cytometry revealed that immobilized, but not soluble MK, significantly promoted the high affinity conformation of ß2 integrins of PMNs. Blocking studies of low-density lipoprotein receptor-related protein 1 (LRP1) suggested that LRP1 may act as a receptor for MK on PMNs. Thus, MK seems to support PMN adhesion by promoting the high affinity conformation of ß2 integrins, thereby facilitating PMN trafficking during acute inflammation.

Two isoforms of Sister-Of-Mammalian Grainyhead have opposing functions in endothelial cells and in vivo.

OBJECTIVE: Sister-of-Mammalian Grainyhead (SOM) is a member of the Grainyhead family of transcription factors. In humans, 3 isoforms are derived from differential first exon usage and alternative splicing and differ only in their N terminal domain. SOM2, the only variant also present in mouse, induces endothelial cell migration and protects against apoptosis. The functions of the human specific isoforms SOM1 and SOM3 have not yet been investigated. Therefore we wanted to elucidate their functions in endothelial cells. APPROACH AND RESULTS: Overexpression of SOM1 in primary human endothelial cells induced migration, phosphorylation of Akt1 and endothelial nitric oxide synthase, and protected against apoptosis, whereas SOM3 had opposite effects; isoform-specific knockdowns confirmed the disparate effects on apoptosis. After reporter assays demonstrated that both are active transcription factors, microarray analyses revealed that they induce different target genes, which could explain the different cellular effects. Overexpression of SOM3 in zebrafish embryos resulted in increased lethality and severe deformations, whereas SOM1 had no deleterious effect. CONCLUSIONS: Our data demonstrate that the splice variant-derived isoforms SOM1 and SOM3 induce opposing effects in primary human endothelial cells and in a whole animal model, most likely through the induction of different target genes.

Hematopoietic progenitor kinase 1 (HPK1) is required for LFA-1-mediated neutrophil recruitment during the acute inflammatory response.

Recruitment of polymorphonuclear neutrophils (PMNs) to sites of acute inflammation critically depends on ß2 integrins (CD11/CD18). Recently, the mammalian actin-binding protein 1 (mAbp1) was identified as an important adaptor protein regulating PMN trafficking downstream of ß2 integrins. Here, we show that mAbp1 constitutively co-immunoprecipitated with hematopoietic progenitor kinase 1 (HPK1) in neutrophil-like differentiated HL-60 (dHL-60) cells. HPK1 was enriched at the lamellipodium of polarized dHL-60 cells, where it colocalized with mAbp1 and actin. Functional analysis of PMNs from HPK1-deficient mice showed that HPK1 was critical for CXCL1-induced lymphocyte function-associated antigen 1 (LFA-1)-mediated PMN adhesion to ICAM-1 under flow conditions. Accordingly, CXCL1-mediated induction of high-affinity LFA-1 required HPK1, but macrophage antigen 1 (Mac-1) affinity regulation was independent of HPK1. Intravital microscopy of the mouse cremaster muscle confirmed the defect of CXCL1-induced leukocyte adhesion in HPK1-deficient mice. Furthermore, ß2 integrin-mediated post-adhesion processes-adhesion strengthening, spreading, and directed mechanotactic crawling of PMNs under flow conditions-involved HPK1 in vitro and in vivo. Upon intrascrotal administration of tumor necrosis factor α (TNF-α), PMN adhesion and extravasation were severely compromised in HPK1-deficient mice. In summary, our results indicate that HPK1 is critically involved in LFA-1-mediated PMN trafficking during acute inflammation.

Unhealthy diet and ultrafine carbon black particles induce senescence and disease associated phenotypic changes.

Diet and pollution are environmental factors known to compromise "healthy aging" of the cardiovascular and respiratory systems. The molecular consequences of this permanent burden in these cells are still unknown. Therefore, this study investigates the impact of unhealthy diet on aging-related signaling pathways of human, primary cardiovascular cells and of airborne particles on lung epithelial and human endothelial cells. Nutrition health reports have shown that the diet in industrialized countries contains more than 100mg/dl low density lipoprotein (LDL) and a high fraction of added sugars, especially fructose. Several studies demonstrated that ultrafine particles can enter the circulation and thus may interact with endothelial cells directly. Both, dietary compounds and pollution derived particles, have been shown to increase the risk for cardiovascular diseases. To simulate an unhealthy diet, we supplemented cell culture media of human primary endothelial cells, smooth muscle cells and cardiomyocytes with LDL and replaced 1/3 of glucose with fructose. We observed hypertrophy in cardiomyocytes, enhanced proliferation in smooth muscle cells and increased senescence, loss of endothelial nitric oxide synthase and increased nuclear FoxO3A in endothelial cells. With respect to pollution we have used ultrafine carbon black particles (ufCB), one of the major constituents of industrial and exhaust emissions, in concentrations our lungs and vessels are constantly exposed to. These concentrations of ufCB increased reactive oxygen species in lung epithelial and vascular endothelial cells and reduced the S-NO content, a marker for NO-bioavailability, in endothelial cells. NO increases activation of Telomerase Reverse Transcriptase (TERT), an enzyme essential for telomere maintenance. TERT is required for proper endothelial cell function and is inactivated by Src kinase under conditions of oxidative stress. ufCB significantly increased Src kinase activation and reduced Telomerase activity in endothelial and lung epithelial cells. As a consequence, ufCB increased senescence of endothelial cells. To investigate whether ufCB show also effects in vivo, we instilled ufCB in concentrations not inducing inflammation into mice. Indeed, eNOS expression was reduced in the abdominal aorta of animals treated with ufCB. Thus, a combination of fructose and LDL in the diet and ufCB, as a major constituent of air pollution, seem to accelerate respiratory and cardiovascular cellular changes, which may compromise "healthy aging" and can lead to cardiovascular and pulmonary diseases.

Carbon nanoparticles induce ceramide- and lipid raft-dependent signalling in lung epithelial cells: a target for a preventive strategy against environmentally-induced lung inflammation.

BACKGROUND: Particulate air pollution in lung epithelial cells induces pathogenic endpoints like proliferation, apoptosis, and pro-inflammatory reactions. The activation of the epidermal growth factor receptor (EGFR) is a key event responsible for signalling events involving mitogen activated protein kinases specific for these endpoints. The molecular events leading to receptor activation however are not well understood. These events are relevant for the toxicological evaluation of inhalable particles as well as for potential preventive strategies in situations when particulate air pollution cannot be avoided. The current study therefore had the objective to elucidate membrane-coupled events leading to EGFR activation and the subsequent signalling cascade in lung epithelial cells. Furthermore, we aimed to identify the molecular target of ectoine, a biophysical active substance which we described to prevent carbon nanoparticle-induced lung inflammation. METHODS: Membrane signalling events were investigated in isolated lipid rafts from lung epithelial cells with regard to lipid and protein content of the signalling platforms. Using positive and negative intervention approaches, lipid raft changes, subsequent signalling events, and lung inflammation were investigated in vitro in lung epithelial cells (RLE-6TN) and in vivo in exposed animals. RESULTS: Carbon nanoparticle treatment specifically led to an accumulation of ceramides in lipid rafts. Detailed analyses demonstrated a causal link of ceramides and subsequent EGFR activation coupled with a loss of the receptor in the lipid raft fractions. In vitro and in vivo investigations demonstrate the relevance of these events for carbon nanoparticle-induced lung inflammation. Moreover, the compatible solute ectoine was able to prevent ceramide-mediated EGFR phosphorylation and subsequent signalling as well as lung inflammation in vivo. CONCLUSION: The data identify a so far unknown event in pro-inflammatory signalling and contribute to the understanding of particle cell interaction and therefore to risk identification and risk assessment of inhalable xenobiotics. Moreover, as this cellular reaction can be prevented by the well tolerated substance ectoine, a molecular preventive strategy for susceptible persons against airway inflammation is proposed.

The mammalian actin-binding protein 1 is critical for spreading and intraluminal crawling of neutrophils under flow conditions.

Recently, the mammalian actin-binding protein 1 (mAbp1; Hip-55, SH3P7, debrin-like protein) was identified as a novel component of the ß(2) integrin-mediated signaling cascade during complement-mediated phagocytosis and firm adhesion of polymorphonuclear neutrophils (PMN) under physiological shear stress conditions. In this study, we found that the genetic ablation of mAbp1 severely compromised not only the induction of adhesion, but also subsequent spreading of leukocytes to the endothelium as assessed by intravital microscopy of inflamed vessels of the cremaster muscle of mice. In vitro studies using murine PMN confirmed that mAbp1 was required for ß(2) integrin-mediated spreading under shear stress conditions, whereas mAbp1 was dispensable for spreading under static conditions. Upon ß(2) integrin-mediated adhesion and chemotactic migration of human neutrophil-like differentiated HL-60 cells, mAbp1 was enriched at the leading edge of the polarized cell. Total internal reflection fluorescence microscopy revealed that mAbp1 formed propagating waves toward the front of the lamellipodium, which are characteristic for dynamic reorganization of the cytoskeleton. Accordingly, binding of mAbp1 to actin was increased upon ß(2) integrin-mediated adhesion, as shown by coimmunoprecipitation experiments. However, chemotactic migration under static conditions was unaffected in the absence of mAbp1. In contrast, the downregulation of mAbp1 by RNA interference technique in neutrophil-like differentiated HL-60 cells or the genetic ablation of mAbp1 in leukocytes led to defective migration under flow conditions in vitro and in inflamed cremaster muscle venules in the situation in vivo. In conclusion, mAbp1 is of fundamental importance for spreading and migration under shear stress conditions, which are critical prerequisites for efficient PMN extravasation during inflammation.

Oxidative stress-induced degradation of thioredoxin-1 and apoptosis is inhibited by thioredoxin-1-actin interaction in endothelial cells.

OBJECTIVE: Thioredoxin-1 (Trx-1), one important antioxidative enzyme in endothelial cells, is required for apoptosis inhibition. Apoptosis induction is dependent on cytoskeletal changes, which depend on actin rearrangements. Therefore, we wanted to elucidate whether a physical interaction exists between Trx-1 and actin and what the functional consequences are. METHODS AND RESULTS: Combined immunoprecipitation/mass spectrometry identified actin as a new binding partner for Trx-1. A separate pool of Trx-1 forms a complex with apoptosis signaling kinase 1. Actin is required for stress fiber formation; thus, the interaction of actin with Trx-1 might interfere with this process. Stress fiber formation, which is directly linked to the phosphorylation of focal adhesion kinase (FAK), occurs as early as 1 hour after H(2)O(2) treatment. It is inhibited by Trx-1 overexpression, treatment with exogenous Trx-1, or inhibition of FAK. Prolonged incubation with H(2)O(2) induced stress fiber formation, reduced Trx-1 protein levels, and increased apoptosis. All these processes were inhibited by preincubation with the FAK inhibitor PF573228. On the contrary, incubation with PF573228 1 hour after H(2)O(2) treatment did not block stress fiber formation, degradation of Trx-1, or apoptosis. CONCLUSIONS: These data demonstrate that the actin-Trx-1 complex protects Trx-1 from degradation and, thus, endothelial cells from apoptosis. Reciprocally, Trx-1 prevents stress fiber formation.

Neutrophil functions and autoimmune arthritis in the absence of p190RhoGAP: generation and analysis of a novel null mutation in mice.

Beta(2) integrins of neutrophils play a critical role in innate immune defense, but they also participate in tissue destruction during autoimmune inflammation. p190RhoGAP (ArhGAP35), a regulator of Rho family small GTPases, is required for integrin signal transduction in fibroblasts. Prior studies have also suggested a role for p190RhoGAP in beta(2) integrin signaling in neutrophils. To directly test that possibility, we have generated a novel targeted mutation completely disrupting the p190RhoGAP-encoding gene in mice. p190RhoGAP deficiency led to perinatal lethality and defective neural development, precluding the analysis of neutrophil functions in adult p190RhoGAP(-/-) animals. This was overcome by transplantation of fetal liver cells from p190RhoGAP(-/-) fetuses into lethally irradiated wild-type recipients. Neutrophils from such p190RhoGAP(-/-) bone marrow chimeras developed normally and expressed normal levels of various cell surface receptors. Although p190RhoGAP(-/-) neutrophils showed moderate reduction of beta(2) integrin-mediated adherent activation, they showed mostly normal migration in beta(2) integrin-dependent in vitro and in vivo assays and normal beta(2) integrin-mediated killing of serum-opsonized Staphylococcus aureus and Escherichia coli. A neutrophil- and beta(2) integrin-dependent transgenic model of the effector phase of autoimmune arthritis also proceeded normally in p190RhoGAP(-/-) bone marrow chimeras. In contrast, all the above responses were completely blocked in CD18(-/-) neutrophils or CD18(-/-) bone marrow chimeras. These results suggest that p190RhoGAP likely does not play a major indispensable role in beta(2) integrin-mediated in vitro and in vivo neutrophil functions or the effector phase of experimental autoimmune arthritis.

Well-known signaling proteins exert new functions in the nucleus and mitochondria.

One distinguishing feature of eukaryotic cells is their compartmentalization into organelles, which all have a unique structural and functional identity. Some proteins are exclusively localized in a single organelle, whereas others are found in more than one. A few proteins, whose function was thought to be completely understood, were only recently found to be present in the mitochondria. Although these proteins come from diverse functional classes, their common new denominator is the regulation of respiratory chain activity. Therefore, this review focuses on new functions of the Signal Transducer and Activator of Transcription 3, originally described as a transcription factor, the most prominent Src kinase family members, Src, Fyn, and Yes, which were so far known as plasma membrane-associated molecular effectors of a variety of extracellular stimuli, the tyrosine phosphatase Shp-2 previously characterized as a modulator of cytosolic signal transduction involved in cell growth, development, inflammation, and chemotaxis, and Telomerase Reverse Transcriptase, the key enzyme preventing telomere erosion in the nucleus. Their unexpected localization in other organelles and regulation of mitochondrial and/or nuclear functions by them adds a new layer of regulatory complexity. This extends the flexibility to cope with changing environmental demands using a limited number of genes and proteins.

Nuclear redox signaling.

Reactive oxygen species have been described to modulate proteins within the cell, a process called redox regulation. However, the importance of compartment-specific redox regulation has been neglected for a long time. In the early 1980s and 1990s, many in vitro studies introduced the possibility that nuclear redox signaling exists. However, the functional relevance for that has been greatly disregarded. Recently, it has become evident that nuclear redox signaling is indeed one important signaling mechanism regulating a variety of cellular functions. Transcription factors, and even kinases and phosphatases, have been described to be redox regulated in the nucleus. This review describes several of these proteins in closer detail and explains their functions resulting from nuclear localization and redox regulation. Moreover, the redox state of the nucleus and several important nuclear redox regulators [Thioredoxin-1 (Trx-1), Glutaredoxins (Grxs), Peroxiredoxins (Prxs), and APEX nuclease (multifunctional DNA-repair enzyme) 1 (APEX1)] are introduced more precisely, and their necessity for regulation of transcription factors is emphasized.

Mitochondrial telomerase reverse transcriptase binds to and protects mitochondrial DNA and function from damage.

OBJECTIVE: The enzyme telomerase and its catalytic subunit the telomerase reverse transcriptase (TERT) are important for maintenance of telomere length in the nucleus. Recent studies provided evidence for a mitochondrial localization of TERT. Therefore, we investigated the exact localization of TERT within the mitochondria and its function. METHODS AND RESULTS: Here, we demonstrate that TERT is localized in the matrix of the mitochondria. TERT binds to mitochondrial DNA at the coding regions for ND1 and ND2. Binding of TERT to mitochondrial DNA protects against ethidium bromide-induced damage. TERT increases overall respiratory chain activity, which is most pronounced at complex I and dependent on the reverse transcriptase activity of the enzyme. Moreover, mitochondrial reactive oxygen species are increased after genetic ablation of TERT by shRNA. Mitochondrially targeted TERT and not wild-type TERT revealed the most prominent protective effect on H(2)O(2)-induced apoptosis. Lung fibroblasts from 6-month-old TERT(-/-) mice (F2 generation) showed increased sensitivity toward UVB radiation and heart mitochondria exhibited significantly reduced respiratory chain activity already under basal conditions, demonstrating the protective function of TERT in vivo. CONCLUSIONS: Mitochondrial TERT exerts a novel protective function by binding to mitochondrial DNA, increasing respiratory chain activity and protecting against oxidative stress-induced damage.

"Shping 2" different cellular localizations - a potential new player in aging processes.

The functions of the ubiquitously expressed protein tyrosine phosphatase Shp-2 are dependent on its localization. Cytosolic Shp-2 is known to modulate different pathways involved in cell growth, cell development, tissue inflammation and cellular chemotaxis. But Shp-2 is also localized in the nucleus and the mitochondria. Nuclear Shp-2 forms a complex with the signal transducer and activator of transcription 5 (STAT5) which then binds to DNA and regulates transcription of milk genes. In contrast, nuclear Shp-2 dephosphorylates STAT1 and thereby inhibits gene transcription. In addition, it counteracts the oxidative stress dependent nuclear export of Telomerase Reverse Transcriptase (TERT) mediated by members of the Src kinase family, a process leading to replicative senescence. For the recently found mitochondrial Shp-2 an involvement in the regulation of the cellular redox balance is discussed. Shp-2 shows the ability to regulate reactive oxygen species formation in the mitochondria. There are hints that mitochondrial Shp-2 and Src are involved in the regulation of respiratory chain activity. Since a substantial fraction of TERT has been found in the mitochondria, it is hypothesized that mitochondrial Shp-2 acts as a positive regulator of TERT in the mitochondria, similar to its nuclear role. Taken together, Shp-2 seems to be a new player in aging processes.

Nuclear protein tyrosine phosphatase Shp-2 is one important negative regulator of nuclear export of telomerase reverse transcriptase.

Aging is one major risk factor for numerous diseases. The enzyme telomerase reverse transcriptase (TERT) plays an important role for aging and apoptosis. Previously, we demonstrated that inhibition of oxidative stress-induced Src kinase family-dependent nuclear export of TERT results in delayed replicative senescence and reduced apoptosis sensitivity. Therefore, the aim of this study was to investigate mechanisms inhibiting nuclear export of TERT. First, we demonstrated that H2O2-induced nuclear export of TERT was abolished in Src, Fyn, and Yes-deficient embryonic fibroblasts. Next, we wanted to identify one potential negative regulator of this export process. One candidate is the protein tyrosine phosphatase Shp-2 (Shp-2), which can counteract activities of the Src kinase family. Indeed, Shp-2 was evenly distributed between the nucleus and cytosol. Nuclear Shp-2 associates with TERT in endothelial cells and dissociates from TERT prior to its nuclear export. Overexpression of Shp-2 wt inhibited H2O2-induced export of TERT. Overexpression of the catalytically inactive, dominant negative Shp-2 mutant (Shp-2(C459S)) reduced endogenous as well as overexpressed nuclear TERT protein and telomerase activity, whereas it had no influence on TERT(Y707F). Binding of TERT(Y707F) to Shp-2 is reduced compared with TERTwt. Ablation of Shp-2 expression led only to an increased tyrosine phosphorylation of TERTwt, but not of TERT(Y707F). Moreover, reduced Shp-2 expression decreased nuclear telomerase activity, whereas nuclear telomerase activity was increased in Shp-2-overexpressing endothelial cells. In conclusion, Shp-2 retains TERT in the nucleus by regulating tyrosine 707 phosphorylation.