Shelterin Telomere Protection Protein 1 Reduction Causes Telomere Attrition and Cellular Senescence via Sirtuin 1 Deacetylase in Chronic Obstructive Pulmonary Disease.

Lung cellular senescence and inflammatory response are the key events in the pathogenesis of chronic obstructive pulmonary disease (COPD) when cigarette smoke (CS) is the main etiological factor. Telomere dysfunction is induced by either critical shortening or disruption of the shelterin complex, leading to cellular senescence. However, it remains unknown whether disruption of the shelterin complex is responsible for CS-induced lung cellular senescence. Here we show that telomere protection protein 1 (TPP1) levels are reduced on telomeres in lungs from mice with emphysema, as well as in lungs from smokers and from patients with COPD. This is associated with persistent telomeric DNA damage, leading to cellular senescence. CS disrupts the interaction of TPP1 with the Sirtuin 1 (Sirt1) complex, leading to increased TPP1 acetylation and degradation. Lung fibroblasts deficient in Sirt1 or treated with a selective Sirt1 inhibitor exhibit increased cellular senescence and decreased TPP1 levels, whereas Sirt1 overexpression and pharmacological activation protect against CS-induced TPP1 reduction and telomeric DNA damage. Our findings support an essential role of TPP1 in protecting CS-induced telomeric DNA damage and cellular senescence, and therefore provide a rationale for a potential therapy for COPD, on the basis of the shelterin complex, in attenuating cellular senescence.

Impaired mitophagy leads to cigarette smoke stress-induced cellular senescence: implications for chronic obstructive pulmonary disease.

Cigarette smoke (CS)-induced cellular senescence is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The molecular mechanism by which CS induces cellular senescence is unknown. Here, we show that CS stress (exposure of primary lung cells to CS extract 0.2-0.75% with a half-maximal inhibitory concentration of ∼0.5%) led to impaired mitophagy and perinuclear accumulation of damaged mitochondria associated with cellular senescence in both human lung fibroblasts and small airway epithelial cells (SAECs). Impaired mitophagy was attributed to reduced Parkin translocation to damaged mitochondria, which was due to CS-induced cytoplasmic p53 accumulation and its interaction with Parkin. Impaired Parkin translocation to damaged mitochondria was also observed in mouse lungs with emphysema (6 months CS exposure, 100 mg TPM/m(3)) as well as in lungs of chronic smokers and patients with COPD. Primary SAECs from patients with COPD also exhibited impaired mitophagy and increased cellular senescence via suborganellar signaling. Mitochondria-targeted antioxidant (Mito-Tempo) restored impaired mitophagy, decreased mitochondrial mass accumulation, and delayed cellular senescence in Parkin-overexpressing cells. In conclusion, defective mitophagy leads to CS stress-induced lung cellular senescence, and restoring mitophagy delays cellular senescence, which provides a promising therapeutic intervention in chronic airway diseases.

Regulation of cytokinesis and its clinical significance.

Dysregulation of the cell cycle leads to polyploid cells, which are classified into mononuclear or binuclear polyploid cells depending on the number of nuclei. Polyploidy is common in plants and in animals. Physiologically, polyploidy and binucleation are differentiation markers and also features of the aging process. In fact, although they provide multiple copies of genes required for survival, a negative correlation between growth capacity and polyploidy has been reported, and thus, suppression or reversal of this phenomenon may be a growth advantage. On the other hand, unscheduled polyploidization may cause genomic instability that might lead to neoplastic aneuploidy. The aim of this review is to analyze the mechanisms that lead to polyploidy, and particularly binucleation, and highlight the potential of ploidy as a marker of illness severity or the success of the adaptive response for an injury, with special emphasis in the liver under physiological and pathological conditions. Hepatocyte binucleation occurs in late fetal development and postnatal maturation, especially after weaning via phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt). It also increases upon aging of the liver as well as in liver cirrhosis and cancer. Liver binucleation mainly indicates the severity of the damage. Furthermore, the eventual increase in hepatocyte binucleation points out compensatory proliferation associated with liver injury. Ploidy conveyor would also permit hepatocyte adaptation to xenobiotic or nutritional injury. In contrast, polyploidy is a feature of many human cancers, and it may predispose to genomic instability and generation of aneuploidization that play a major role in carcinogenesis. Finally, a better understanding of the polyploidization process is needed in order to approach clinical research but also, to get deeper knowledge of cell cycle control. The fascinating regulation of cell cycle in liver and the generation and reversal of ploidies will provide more clues for the mystery of liver regeneration.

Liver-specific p38α deficiency causes reduced cell growth and cytokinesis failure during chronic biliary cirrhosis in mice.

UNLABELLED: p38α mitogen-activated protein kinases (MAPK) may be essential in the up-regulation of proinflammatory cytokines and can be activated by transforming growth factor ß, tumor necrosis factor-α, interleukin-1ß, and oxidative stress. p38 MAPK activation results in hepatocyte growth arrest, whereas increased proliferation has been considered a hallmark of p38α-deficient cells. Our aim was to assess the role of p38α in the progression of biliary cirrhosis induced by chronic cholestasis as an experimental model of chronic inflammation associated with hepatocyte proliferation, apoptosis, oxidative stress, and fibrogenesis. Cholestasis was induced in wildtype and liver-specific p38α knockout mice by bile duct ligation and animals were sacrificed at 12 and 28 days. p38α knockout mice exhibited a 50% decrease in mean life-span after cholestasis induction. MK2 phosphorylation was markedly reduced in liver of p38α-deficient mice upon chronic cholestasis. Hepatocyte growth was reduced and hepatomegaly was absent in p38α-deficient mice during chronic cholestasis through down-regulation of both AKT and mammalian target of rapamycin. Cyclin D1 and cyclin B1 were up-regulated in liver of p38α-deficient mice upon chronic cholestasis, but unexpectedly proliferating cell nuclear antigen was down-regulated at 12 days after cholestasis induction and the mitotic index was very high upon cholestasis in p38α-deficient mice. p38α-knockout hepatocytes exhibited cytokinesis failure evidenced by an enhanced binucleation rate. As chronic cholestasis evolved the binucleation rate decreased in wildtype animals, whereas it remained high in p38α-deficient mice. CONCLUSION: Our results highlight a key role of p38α in hepatocyte proliferation, in the development of hepatomegaly, and in survival during chronic inflammation such as biliary cirrhosis.

Mitochondrial biogenesis fails in secondary biliary cirrhosis in rats leading to mitochondrial DNA depletion and deletions.

Chronic cholestasis is characterized by mitochondrial dysfunction, associated with loss of mitochondrial membrane potential, decreased activities of respiratory chain complexes, and ATP production. Our aim was to determine the molecular mechanisms that link long-term cholestasis to mitochondrial dysfunction. We studied a model of chronic cholestasis induced by bile duct ligation in rats. Key sensors and regulators of the energetic state and mitochondrial biogenesis, mitochondrial DNA (mtDNA)-to-nuclear DNA (nDNA) ratio (mtDNA/nDNA) relative copy number, mtDNA deletions, and indexes of apoptosis (BAX, BCL-2, and cleaved caspase 3) and cell proliferation (PCNA) were evaluated. Our results show that long-term cholestasis is associated with absence of activation of key sensors of the energetic state, evidenced by decreased SIRT1 and pyruvate dehydrogenase kinase levels and lack of AMPK activation. Key mitochondrial biogenesis regulators (PGC-1α and GABP-α) decreased and NRF-1 was not transcriptionally active. Mitochondrial transcription factor A (TFAM) protein levels increased transiently in liver mitochondria at 2 wk after bile duct ligation, but they dramatically decreased at 4 wk. Reduced TFAM levels at this stage were mirrored by a marked decrease (65%) in mtDNA/nDNA relative copy number. The blockade of mitochondrial biogenesis should not be ascribed to activation of apoptosis or inhibition of cell proliferation. Impaired mitochondrial turnover and loss of the DNA stabilizing effect of TFAM are likely the causative event involved in the genetic instability evidenced by accumulation of mtDNA deletions. In conclusion, the lack of stimulation of mitochondrial biogenesis leads to mtDNA severe depletion and deletions in long-term cholestasis. Hence, long-term cholestasis should be considered a secondary mitochondrial hepatopathy.

p38α deficiency restrains liver regeneration after partial hepatectomy triggering oxidative stress and liver injury.

p38α MAPK negatively regulates the G1/S and G2/M cell cycle transitions. However, liver-specific p38α deficiency impairs cytokinesis and reduces hepatocyte proliferation during cirrhosis and aging in mice. In this work, we have studied how p38α down-regulation affects hepatocyte proliferation after partial hepatectomy, focusing on mitotic progression, cytokinesis and oxidative stress. We found that p38α deficiency triggered up-regulation of cyclins A1, B1, B2, and D1 under basal conditions and after hepatectomy. Moreover, p38α-deficient hepatocytes showed enhanced binucleation and increased levels of phospho-histone H3 but impaired phosphorylation of MNK1 after hepatectomy. The recovery of liver mass was transiently delayed in mice with p38α-deficient hepatocytes vs wild type mice. We also found that p38α deficiency caused glutathione oxidation in the liver, increased plasma aminotransferases and lactate dehydrogenase activities, and decreased plasma protein levels after hepatectomy. Interestingly, p38α silencing in isolated hepatocytes markedly decreased phospho-MNK1 levels, and silencing of either p38α or Mnk1 enhanced binucleation of hepatocytes in culture. In conclusion, p38α deficiency impairs mitotic progression in hepatocytes and restrains the recovery of liver mass after partial hepatectomy. Our results also indicate that p38α regulates cytokinesis by activating MNK1 and redox modulation.

Age-dependent regulation of antioxidant genes by p38α MAPK in the liver.

p38α is a redox sensitive MAPK activated by pro-inflammatory cytokines and environmental, genotoxic and endoplasmic reticulum stresses. The aim of this work was to assess whether p38α controls the antioxidant defense in the liver, and if so, to elucidate the mechanism(s) involved and the age-related changes. For this purpose, we used liver-specific p38α-deficient mice at two different ages: young-mice (4 months-old) and old-mice (24 months-old). The liver of young p38α knock-out mice exhibited a decrease in GSH levels and an increase in GSSG/GSH ratio and malondialdehyde levels. However, old mice deficient in p38α had higher hepatic GSH levels and lower GSSG/GSH ratio than young p38α knock-out mice. Liver-specific p38α deficiency triggered a dramatic down-regulation of the mRNAs of the key antioxidant enzymes glutamate cysteine ligase, superoxide dismutase 1, superoxide dismutase 2, and catalase in young mice, which seems mediated by the lack of p65 recruitment to their promoters. Nrf-2 nuclear levels did not change significantly in the liver of young mice upon p38α deficiency, but nuclear levels of phospho-p65 and PGC-1α decreased in these mice. p38α-dependent activation of NF-κB seems to occur through classical IκB Kinase and via ribosomal S6 kinase1 and AKT in young mice. However, unexpectedly the long-term deficiency in p38α triggers a compensatory up-regulation of antioxidant enzymes via NF-κB activation and recruitment of p65 to their promoters. In conclusion, p38α MAPK maintains the expression of antioxidant genes in liver of young animals via NF-κΒ under basal conditions, whereas its long-term deficiency triggers compensatory up-regulation of antioxidant enzymes through NF-κΒ.

p38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and aging.

BACKGROUND: Hepatocyte poliploidization is an age-dependent process, being cytokinesis failure the main mechanism of polyploid hepatocyte formation. Our aim was to study the role of p38α MAPK in the regulation of actin cytoskeleton and cytokinesis in hepatocytes during development and aging. METHODS: Wild type and p38α liver-specific knock out mice at different ages (after weaning, adults and old) were used. RESULTS: We show that p38α MAPK deficiency induces actin disassembly upon aging and also cytokinesis failure leading to enhanced binucleation. Although the steady state levels of cyclin D1 in wild type and p38α knock out old livers remained unaffected, cyclin B1- a marker for G2/M transition- was significantly overexpressed in p38α knock out mice. Our findings suggest that hepatocytes do enter into S phase but they do not complete cell division upon p38α deficiency leading to cytokinesis failure and binucleation. Moreover, old liver-specific p38α MAPK knock out mice exhibited reduced F-actin polymerization and a dramatic loss of actin cytoskeleton. This was associated with abnormal hyperactivation of RhoA and Cdc42 GTPases. Long-term p38α deficiency drives to inactivation of HSP27, which seems to account for the impairment in actin cytoskeleton as Hsp27-silencing decreased the number and length of actin filaments in isolated hepatocytes. CONCLUSIONS: p38α MAPK is essential for actin dynamics with age in hepatocytes.

Long term p38-a deficiency up-regulates antioxidant enzymes through compensatory NF-?B activation.

p38a MAPK may function as a mediator of reactive oxygen species signaling and thus p38a is considered a sensor of oxidative stress. In liver-specific p38a knock-out (KO) adult mice we previously found glutathione depletion and down-regulation of antioxidant enzymes. Our aim was to assess the influence of long-term p38a deficiency on oxidative stress and on the regulation of antioxidant enzymes in liver of old mice. To this end, wild type or liver-specific KO mice after weaning, at 4-6 months of age, or at 24 months of age were used. Reduced glutathione (GSH) and oxidized glutathione levels were determined by mass spectrometry, gene expression of antioxidant enzymes was determined by RT-PCR, and induction of NRF-2 and PGC-1a as well as activation of NF-?B were assessed by western blotting. We report that GSH levels decreased upon aging only in liver of wild-type mice, but not in p38a KO mice. The mRNA expression of glutathione peroxidase, Cu-Zn superoxide dismutase, Mn-superoxide dismutase, and glutamate cysteine ligase was up-regulated in adult wild-type in comparison with mice after weaning, but their gene expression was down-regulated in old wild-type mice. Although the mRNA expression of glutathione peroxidase, Cu-Zn superoxide dismutase, Mn-superoxide dismutase, and glutamate cysteine ligase was down-regulated in adult KO mice vs KO mice after weaning, their gene expression was up-regulated in old KO mice. This up-regulation was not associated with nuclear translocation of NRF-2, which decreased upon aging in KO mice, nor with up-regulation of PGC-1a. However, phosphorylation of p65 was markedly increased in old KO mice as an index of NF-?B activation. In conclusion, long term deficiency of p38a in the liver causes compensatory activation of NF?B that is likely to be responsible for the up-regulation of antioxidant enzymes upon aging, independently of Nrf-2 and PGC-1a.

p38α deficiency and oxidative stress cause cytokinesis failure in hepatocytes.

Cytokinesis is the last step in mitosis and it implies re-organization of the actin cytoskeleton. Its failure is one of the major mechanisms of polyploidy and binucleation in mammals. Our aims were 1) to assess the role of redox-sensitive p38α MAPK in cytokinesis by studying the liver of wild type mice or liver-specific p38α knock-out mice; 2) to assess the role of oxidative stress associated with hepatocyte isolation on cytokinesis. When p38α was down-regulated in hepatocytes, MK2 phosphorylation on threonine 334 was completely abrogated. Activation of MNK-1, required for abscission of the intercellular bridge, was diminished. Key proteins of the RhoA pathway (phospho-PRK2, nuclear phosphorylated cofilin, and cytosolic p27) were assessed confirming the impairment of this pathway. The absence of p38α in aging liver also led to a decrease in HSP27 phosphorylation, which is required for actin polymerization. Indeed, a severe impairment in the F-actin filamentous structure was found in the liver of old mice upon p38α deficiency. Consequently, long-term p38α MAPK down-regulation markedly affects the RhoA pathway and actin cytoskeleton dynamics inducing actin disassembly and cytokinesis failure upon aging. On the other hand, hepatocyte isolation caused marked glutathione depletion, increased generation of reactive oxygen species, and activated cell cycle entry. Addition of N-acetyl cysteine to isolation media prevented glutathione depletion, restrained the cell cycle entry, and abrogated defective cytokinesis and the formation of binucleated hepatocytes during isolation. Our results show that hepatocytes do enter into S phase but they do not complete cell division with age upon p38α deficiency or upon oxidative stress associated with isolation leading in both cases to cytokinesis failure and binucleation.

Mitochondrial dysfunction in cholestatic liver diseases.

Cholestatic liver diseases are characterized by blockade of bile flow from the liver to the intestine, and accumulation of hydrophobic bile acids in the liver and plasma. As a consequence an inflammatory response evolves associated with increased apoptosis, oxidative stress, and eventually fibrosis. Cholestasis is associated with profound metabolic changes, alterations in the mitochondrial function, decreased fatty acid oxidation, and increased glycolisis. Mitochondria play a central role in the development of this liver disease because they mediate death receptor signaling - triggered by inflammatory cytokines or bile acids - and contribute to oxidative damage, metabolic disorder, and onset of fibrosis. During the pathogenesis of biliary cirrhosis mitochondria's need for renewal is hampered by a blunted mitochondrial biogenesis. Lack of stimulation of mitochondrial renewal helps to explain mitochondrial impairment in long-term cholestasis. The marked depletion of mitochondrial DNA and occurrence of mitochondrial DNA deletions are probably relevant contributors to the progression of this severe disease. All these findings certainly support the consideration of long-term cholestasis as a secondary mitochondrial hepatopathy.