Replicative senescence is distinguishable from DNA damage-induced senescence by increased methylation of promoter of rDNA and reduced expression of rRNA.

Human fibroblasts become senescent after a limited number of replications or by diverse stresses, such as DNA damage. However, replicative and damage induced senescence are indistinguishable in respect to proliferation cessation and expression of senescence markers, senescence-associated ß-galactosidase, p16 and p21. Here, we show that senescence types can be distinguished by reduced levels of 18S, 5.8S and 28S rRNA, in replicative but not induced senescence. We also demonstrate that promoter region of rRNA is hypermethylated in replicative senescence. The findings show that expression level of rRNA or methylation of its promoter can be used to distinguish between senescence types.

3-Mercaptopyruvate sulfurtransferase disruption in dermal fibroblasts facilitates adipogenic trans-differentiation.

Transitioning from a differentiated state to a higher-order of plasticity, by partial rather than full reactivation of pluripotency genes, might be a better approach in regenerative medicine. Hydrogen sulfide plays a crucial role in the maintenance and differentiation of mesenchymal stem cells (MSC) that have the potential to differentiate to a diverse group of mesenchymally derived cells. It was shown that these cells show a heavy reliance on cystathionine-ß-synthase (CBS)-derived hydrogen sulfide (H2S) during differentiation. We have found that expression and activity of 3-mercaptopyruvate sulfurtransferase (MPST), one of three enzymes that hat regulates H2S biosynthesis, is significantly lower in MSC as compared with lineage-restricted dermal fibroblasts. Here, we tested the hypothesis that suppression of MPST in dermal fibroblasts might induce plasticity-related changes and broaden the transdifferentiation potency. Inactivation of MPST with phenylpyruvate (PP) or by siRNA silencing led to diminished H2S production associated with increased production of reactive oxygen species (ROS) and lactic acid. Accumulation of α-ketoglutarate (α-KG), a key metabolite required for the expression of ten-eleven translocation hydroxylase (TET), was associated with stimulated transcription of pluripotency related genes including OCT4, KLF4, SOX2, and NANOG. Suppression of TET1 gene and inhibition of glycolysis with glucose analog, 2-desoxy-d-glucose, or hexokinase II inhibitor significantly reduced expression of pluripotency genes following MPST inactivation or knockdown. MPST disruption promoted the conversion of fibroblasts into adipocytes as evidenced by a significant increase in expression of adipocyte-specific genes, PPARγ, and UCP1, and intracellular accumulation of oil Red-O positive fat droplets. Inhibition of glycolysis inhibited these changes. Under induced differentiation conditions, fibroblasts with disrupted MPST show the potency to differentiate to white adipogenic lineage. Thus, MPST inactivation or silencing enhances the plasticity of dermal fibroblasts in a TET1 and glycolysis dependent manner and promotes adipogenic transdifferentiation.

Replicative Senescence in Human Fibroblasts Is Delayed by Hydrogen Sulfide in a NAMPT/SIRT1 Dependent Manner.

Recent evidence suggests that hydrogen sulfide (H2S) has cytoprotective and anti-aging effects. However, the mechanisms for such properties are not fully understood. Here, we show that the expression of the main H2S producing enzyme, CBS, and production of H2S are coordinately diminished in replicative senescent adult human dermal fibroblasts. The reduced production of H2S falls within the same time-frame that the hallmarks of replicative senescence appear including accumulation of SA-ß-Gal, enhanced expression of p16, p21, and RRM2B while the expression of RRM2, hTERT, SIRT1, NAMPT, and NAD/NADH ratio all fall. Exogenous H2S increases the expression of hTERT, NAMPT, SIRT1 and NAD/NADH ratio in treated cells. Moreover, H2S safeguards the expression of hTERT in a NAMPT and SIRT1 dependent manner and delays the onset of replicative senescence as evidenced by reduced accumulation of age associated SA-ß-Gal and cessation of proliferation. Postponement of loss of cell proliferative capacity without risk of mutagenesis shows implications for use of H2S in delaying the adverse effects of senescence in organisms.

Dedifferentiation of cancer cells following recovery from a potentially lethal damage is mediated by H2S-Nampt.

Recently, we reported that cancer cells that recover from a potentially lethal damage gain new phenotypic features comprised of mitochondrial structural remodeling associated with increased glycolytic dependency and drug resistance. Here, we demonstrate that a subset of cancer cells, upon recovery from a potentially lethal damage, undergo dedifferentiation and express genes, which are characteristic of undifferentiated stem cells. While these cells are competent in maintaining differentiated progeny of tumor, they also exhibit transdifferentiation potential. Dedifferentiation is characterized by accumulation of hydrogen sulfide (H2S), which triggers up-regulation of nicotinamide phosphoribosyltransferase (Nampt) accompanied by changes in the redox state. The molecular events triggered by Nampt include elevated production of NAD(+) and up-regulation of H2S producing enzymes, cystathionine beta synthase (CBS) and cystathionase (CTH) with 3-mercaptopyruvate sulfurtransferase (MST) being detectable only in 3D spheroids. Suppression of Nampt, or inactivation of H2S producing enzymes, all reduce H2S production and reverse the ability of cells to dedifferentiate. Moreover, H2S induced stem cell markers in parental cancer cells in a manner similar to that observed in damage recovered cells. These data suggest of existence of a positive feedback loop between H2S and Nampt that controls dedifferentiation in cancer cells that recover from a potentially lethal damage.

A H2S-Nampt dependent energetic circuit is critical to survival and cytoprotection from damage in cancer cells.

We recently demonstrated that cancer cells that recover from damage exhibit increased aerobic glycolysis, however, the molecular mechanism by which cancer cells survive the damage and show increased aerobic glycolysis remains unknown. Here, we demonstrate that diverse cancer cells that survive hypoxic or oxidative damage show rapid cell proliferation, and develop tolerance to damage associated with increased production of hydrogen sulfide (H2S) which drives up-regulation of nicotinamide phosphoribosyltransferase (Nampt). Consistent with existence of a H2S-Nampt energetic circuit, in damage recovered cancer cells, H2S, Nampt and ATP production exhibit a significant correlation. Moreover, the treatment of cancer cells with H2S donor, NaHS, coordinately increases Nampt and ATP levels, and protects cells from drug induced damage. Inhibition of cystathionine beta synthase (CBS) or cystathionase (CTH), enzymes which drive generation of H2S, decreases Nampt production while suppression of Nampt pathway by FK866, decreases H2S and ATP levels. Damage recovered cells isolated from tumors grown subcutaneously in athymic mice also show increased production of H2S, Nampt and ATP levels, associated with increased glycolysis and rapid proliferation. Together, these data show that upon recovery from potential lethal damage, H2S-Nampt directs energy expenditure and aerobic glycolysis in cancer cells, leads to their exponential growth, and causes a high degree of tolerance to damage. Identification of H2S-Nampt as a pathway responsible for induction of damage tolerance in cancer cells may underlie resistance to therapy and offers the opportunity to target this pathway as a means in treatment of cancer.

Cancer cells recovering from damage exhibit mitochondrial restructuring and increased aerobic glycolysis.

Instead of relying on mitochondrial oxidative phosphorylation, most cancer cells rely heavily on aerobic glycolysis, a phenomenon termed as "the Warburg effect". We considered that this effect is a direct consequence of damage which persists in cancer cells that recover from damage. To this end, we studied glycolysis and rate of cell proliferation in cancer cells that recovered from severe damage. We show that in vitro Damage-Recovered (DR) cells exhibit mitochondrial structural remodeling, display Warburg effect, and show increased in vitro and in vivo proliferation and tolerance to damage. To test whether cancer cells derived from tumor microenvironment can show similar properties, we isolated Damage-Recovered (T(DR)) cells from tumors. We demonstrate that T(DR) cells also show increased aerobic glycolysis and a high proliferation rate. These findings show that Warburg effect and its consequences are induced in cancer cells that survive severe damage.

The Shigella OspC3 effector inhibits caspase-4, antagonizes inflammatory cell death, and promotes epithelial infection.

Caspase-mediated inflammatory cell death acts as an intrinsic defense mechanism against infection. Bacterial pathogens deploy countermeasures against inflammatory cell death, but the mechanisms by which they do this remain largely unclear. In a screen for Shigella flexneri effectors that regulate cell death during infection, we discovered that Shigella infection induced acute inflammatory, caspase-4-dependent epithelial cell death, which is counteracted by the bacterial OspC3 effector. OspC3 interacts with the caspase-4-p19 subunit and inhibits its activation by preventing caspase-4-p19 and caspase-4-p10 heterodimerization by depositing the conserved OspC3 X1-Y-X2-D-X3 motif at the putative catalytic pocket of caspase-4. Infection of guinea pigs with a Shigella ospC3-deficient mutant resulted in enhanced inflammatory cell death and associated symptoms, correlating with decreased bacterial burdens. Salmonella Typhimurium and enteropathogenic Escherichia coli infection also induced caspase-4-dependent epithelial death. These findings highlight the importance of caspase-4-dependent innate immune responses and demonstrate that Shigella delivers a caspase-4-specific inhibitor to delay epithelial cell death and promote infection.

The endocytic recycling regulator EHD1 is essential for spermatogenesis and male fertility in mice.

BACKGROUND: The C-terminal Eps15 homology domain-containing protein 1 (EHD1) is ubiquitously expressed and regulates the endocytic trafficking and recycling of membrane components and several transmembrane receptors. To elucidate the function of EHD1 in mammalian development, we generated Ehd1-/- mice using a Cre/loxP system. RESULTS: Both male and female Ehd1-/- mice survived at sub-Mendelian ratios. A proportion of Ehd1-/- mice were viable and showed smaller size at birth, which continued into adulthood. Ehd1-/- adult males were infertile and displayed decreased testis size, whereas Ehd1-/- females were fertile. In situ hybridization and immunohistochemistry of developing wildtype mouse testes revealed EHD1 expression in most cells of the seminiferous epithelia. Histopathology revealed abnormal spermatogenesis in the seminiferous tubules and the absence of mature spermatozoa in the epididymides of Ehd1-/- males. Seminiferous tubules showed disruption of the normal spermatogenic cycle with abnormal acrosomal development on round spermatids, clumping of acrosomes, misaligned spermatids and the absence of normal elongated spermatids in Ehd1-/- males. Light and electron microscopy analyses indicated that elongated spermatids were abnormally phagocytosed by Sertoli cells in Ehd1-/- mice. CONCLUSIONS: Contrary to a previous report, these results demonstrate an important role for EHD1 in pre- and post-natal development with a specific role in spermatogenesis.

Control of Alzheimer's amyloid beta toxicity by the high molecular weight immunophilin FKBP52 and copper homeostasis in Drosophila.

FK506 binding proteins (FKBPs), also called immunophilins, are prolyl-isomerases (PPIases) that participate in a wide variety of cellular functions including hormone signaling and protein folding. Recent studies indicate that proteins that contain PPIase activity can also alter the processing of Alzheimer's Amyloid Precursor Protein (APP). Originally identified in hematopoietic cells, FKBP52 is much more abundantly expressed in neurons, including the hippocampus, frontal cortex, and basal ganglia. Given the fact that the high molecular weight immunophilin FKBP52 is highly expressed in CNS regions susceptible to Alzheimer's, we investigated its role in Abeta toxicity. Towards this goal, we generated Abeta transgenic Drosophila that harbor gain of function or loss of function mutations of FKBP52. FKBP52 overexpression reduced the toxicity of Abeta and increased lifespan in Abeta flies, whereas loss of function of FKBP52 exacerbated these Abeta phenotypes. Interestingly, the Abeta pathology was enhanced by mutations in the copper transporters Atox1, which interacts with FKBP52, and Ctr1A and was suppressed in FKBP52 mutant flies raised on a copper chelator diet. Using mammalian cultures, we show that FKBP52 (-/-) cells have increased intracellular copper and higher levels of Abeta. This effect is reversed by reconstitution of FKBP52. Finally, we also found that FKBP52 formed stable complexes with APP through its FK506 interacting domain. Taken together, these studies identify a novel role for FKBP52 in modulating toxicity of Abeta peptides.

Autophosphorylation docking site Tyr-867 in Mer receptor tyrosine kinase allows for dissociation of multiple signaling pathways for phagocytosis of apoptotic cells and down-modulation of lipopolysaccharide-inducible NF-kappaB transcriptional activation.

Efficient clearance of apoptotic cells is essential for tissue homeostasis, allowing for cellular turnover without inflammatory consequences. The Mer (Nyk and c-Eyk) receptor tyrosine kinase (Mertk) is involved in two aspects of apoptotic cell clearance by acting as a receptor for Gas6, a gamma-carboxylated phosphatidylserine-binding protein that bridges apoptotic and viable cells. First, Mertk acts in a bona fide engulfment pathway in concert with alphavbeta5 integrin by regulating cytoskeletal assemblages, and second, it acts as a negative regulator for inflammation by down-modulating pro-inflammatory signals mediated from bacterial lipopolysaccharide-Toll-like receptor 4 (TLR4) signaling, and hence recapitulating anti-inflammatory immune modulation by apoptotic cells. Here we describe Mertk post-receptor events that govern phagocytosis and cytoskeletal signaling are principally mediated by autophosphorylation site Tyr-867. Using the Mertk Y867F mutant and pharmacological inhibitors, we show that Tyr-867 is required for phosphatidylinositol 3-kinase and phospholipase Cgamma2 activation; their activation in turn elicits protein kinase C-dependent signals that act on the actin cytoskeleton. Although Mertk(Y867F) blocked the tyrosine phosphorylation of FAK on Tyr-861 and p130(cas) and also abrogated the phagocytosis of apoptotic cells, this mutant did not suppress lipopolysaccharide-inducible NF-kappaB transcription, nor was NF-kappaB activation dependent on the protein kinase C inhibitor, calphostin C. Finally, unlike the cytoskeletal events associated with Tyr-867 autophosphorylation, the trans-inhibition of NF-kappaB occurred in a postnuclear-dependent fashion independent of cytosolic IkappaB phosphorylation and p65/RelA sequestration. Taken together, these data suggest that Mertk has distinct and separable effects for phagocytosis and for resolving inflammation, providing a molecular rationale for how immune licensing and inflammation can be dissociated from phagocytosis in a single phagocytic receptor.

C-terminal SH3 domain of CrkII regulates the assembly and function of the DOCK180/ELMO Rac-GEF.

Genetic studies in Caenorhabditis elegans identified an evolutionarily conserved CED-2 (CrkII), CED-5 (DOCK180), CED-12 (ELMO), CED-10 (Rac1) module important for cell migration and phagocytosis of apoptotic cells. Previous studies have shown that DOCK180 and ELMO comprise an unconventional bipartite Dbl homology domain-independent Rac guanine nucleotide exchange factor (Rac-GEF); but it is still unclear how CrkII functions in Rac-GEF activity. In this study, we have characterized a unique function of CrkII in phagocytosis and Rac activation mediated by the C-terminal SH3 domain, a region of CrkII that has no clear cellular or biochemical function. We found that mutations that disrupt the C-terminal SH3 domain of CrkII (CrkII-SH3-C) abrogate engulfment of apoptotic cells and impair cell spreading on extracellular matrix. Surprisingly, despite the effects on engulfment, W276K CrkII strongly potentiated Rac-GTP loading when ectopically expressed in HEK 293T cells. Contrary to the effects of the true dominant negative SH2 domain mutants (R38K CrkII) and SH3-N domain mutants (W170K CrkII) that prevent macromolecular assembly of signaling proteins, W276K CrkII increases association between DOCK180 and CrkII as well as constitutive tethering of the Crk/DOCK180/ELMO protein complex that interacted with RhoG. Our results indicate that while N-terminal SH3 of CrkII promotes assembly between CrkII and DOCK180, the C-terminal SH3 of CrkII regulates the stability and turnover of the DOCK180/ELMO complex. Studies with W276K CrkII may offer a unique opportunity to study the structure and function of the DOCK180/ELMO Rac-GEF.

A novel role for the immunophilin FKBP52 in copper transport.

FK506-binding protein 52 (FKBP52) is an immunophilin that possesses peptidylprolyl cis/trans-isomerase (PPIase) activity and is a component of a subclass of steroid hormone receptor complexes. Several recent studies indicate that immunophilins can regulate neuronal survival and nerve regeneration although the molecular mechanisms are poorly understood. To investigate the function of FKBP52 in the nervous system, we employed a yeast two-hybrid strategy using the PPIase domain (domain I) as bait to screen a neonatal rat dorsal root ganglia cDNA expression library. We identified an interaction between FKBP52 domain I and Atox1, a copper-binding metallochaperone. Atox1 interacts with Menkes disease protein and Wilson disease protein (WD) and functions in copper efflux. The interaction between FKBP52 and Atox1 was observed in both glutathione S-transferase pull-down experiments and when proteins were ectopically expressed in human embryonic kidney (HEK) 293T cells and was sensitive to FK506. Interestingly, the FKBP52/Atox1 interaction was enhanced when HEK 293T cells were cultured in copper-supplemented medium and decreased in the presence of the copper chelator, bathocuproine disulfate, suggesting that the interaction is regulated in part by intracellular copper. Overexpression of FKBP52 increased rapid copper efflux in (64)Cu-loaded cells, as did the overexpression of WD transporter. Taken together, our present findings suggest that FKBP52 is a component of the copper efflux machinery, and in so, may also promote neuroprotection from copper toxicity.

The opsonin MFG-E8 is a ligand for the alphavbeta5 integrin and triggers DOCK180-dependent Rac1 activation for the phagocytosis of apoptotic cells.

Opsonization of apoptotic cells facilitates recognition by phagocytes for the rapid clearance of potentially inflammatory cellular material. The secreted glycoprotein Milk Fat Globule Factor-E8 (MFG-E8) is a member of this family of bridging molecules and is believed to bind phosphatidylserine (PS) on the dying cell, linking it to integrin receptors on the phagocyte. Here we report the characterization of a functional signaling module involving MFG-E8, alphavbeta5 integrin, and DOCK180 for the activation of Rac1. We show that MFG-E8 and DOCK180 are both expressed in phagocytic-competent primary immature dendritic cells (DCs) and DC2.4 cells, and are potently down-regulated upon DC maturation, consistent with their role in phagocytosis and antigen capture. Coexpression of MFG-E8 with alphavbeta5 integrin potentiated integrin-mediated Rac1 activation, which was abrogated by mutagenesis in the RGD motif in MFG-E8. Moreover, expression of antisense DOCK180 abrogated MFG-E8-alphavbeta5-mediated Rac activation and impaired the phagocytosis of apoptotic cells. These data demonstrate a biochemical link between an opsonin of apoptotic cells, the alphavbeta5 integrin, and the Crk-DOCK180-Rac1 pathway, and importantly, show that MFG-E8 and DOCK180 are expressed according to the functional status of the phagocyte.

Mutations in the occQ operator that decrease OccR-induced DNA bending do not cause constitutive promoter activity.

OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid. Positive control of the occ genes occurs in response to octopine, a metabolite released from plant tumors. Octopine causes DNA-bound OccR to undergo a conformational change from an inactive to an active state; this change is marked by a decrease in footprint length from 55 to 45 nucleotides as well as a relaxation of a high angle DNA bend. In this study, we first used gel filtration chromatography to show that OccR is dimeric in solution, and we used gel shift assays to show that OccR is tetrameric when bound to DNA. We then created a series of site-directed mutations in the OccR-binding site. Some mutations were designed to lock OccR-DNA complexes into a conformation resembling the inactive conformation, whereas other mutations were designed to lock complexes into the active conformation. These mutations altered the conformation of OccR-DNA complexes and their responses to octopine in ways that we had predicted. As expected, operator mutations that locked complexes into a conformation having a long footprint and a high angle DNA bend blocked activation by octopine in vivo. Surprisingly, however, mutations that lock OccR into a short footprint and low angle DNA bend failed to cause the protein to function constitutively. Furthermore, some of the latter mutations interfered with activation by octopine. We conclude that locking OccR into a conformation having a short footprint is not sufficient to cause constitutive activation, and octopine must cause at least one additional conformational change in the protein.

Constitutive mutations of the OccR regulatory protein affect DNA bending in response to metabolites released from plant tumors.

OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid and is also an autorepressor. Positive control of the occ genes occurs in response to octopine, a nutrient released from crown gall tumors. OccR binds to a site upstream of the occQ promoter in the presence and absence of octopine. Octopine causes prebound OccR to undergo a conformational change at the DNA binding site that causes changes in footprint length and DNA bending. To determine the roles of these conformational changes in transcriptional activation, we isolated 22 OccR mutants that were able to activate the occQ promoter in the absence of octopine. Thirteen of these mutants contained single amino acid substitutions, and nine contained two base pair changes resulting in two amino acid substitutions, which in most cases acted synergistically. These mutations spanned the entire length of the protein. Most of these mutant proteins in the absence of octopine displayed DNA binding and bending properties characteristic of transcriptionally active OccR-octopine complexes.