Mitochondrial Sulfide Quinone Oxidoreductase Prevents Activation of the Unfolded Protein Response in Hydrogen Sulfide

Hydrogen sulfide (H₂S) is an endogenously produced gaseous molecule with important roles in cellular signaling. In mammals, exogenous H₂S improves survival of ischemia/reperfusion. We have previously shown that exposure to H₂S increases the lifespan and thermotolerance in Caenorhabditis elegans, and improves protein homeostasis in low oxygen. The mitochondrial SQRD-1 (sulfide quinone oxidoreductase) protein is a highly conserved enzyme involved in H₂S metabolism. SQRD-1 is generally considered important to detoxify H₂S. Here, we show that SQRD-1 is also required to maintain protein translation in H₂S. In sqrd-1 mutant animals, exposure to H₂S leads to phosphorylation of eIF2α and inhibition of protein synthesis. In contrast, global protein translation is not altered in wild-type animals exposed to lethally high H₂S or in hif-1(ia04) mutants that die when exposed to low H₂S. We demonstrate that both gcn-2 and pek-1 kinases are involved in the H₂S-induced phosphorylation of eIF2α. Both ER and mitochondrial stress responses are activated in sqrd-1 mutant animals exposed to H₂S, but not in wild-type animals. We speculate that SQRD-1 activity in H₂S may coordinate proteostasis responses in multiple cellular compartments.     

Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

ATM and ATR protein kinases play a crucial role in cellular DNA damage responses. The inhibition of ATM and ATR can lead to the abolition of the function of cell cycle checkpoints. In this regard, it is expected that checkpoint inhibitors can serve as sensitizing agents for anti-cancer chemo/radiotherapy. Although several ATM inhibitors have been reported, there are no ATR-specific inhibitors currently available. Here, we report the inhibitory effect of schisandrin B (SchB), an active ingredient of Fructus schisandrae, on ATR activity in DNA damage response. SchB treatment significantly decreased the viability of A549 adenocarcinoma cells after UV exposure. Importantly, SchB treatment inhibited both the phosphorylation levels of ATM and ATR substrates, as well as the activity of the G2/M checkpoint in UV-exposed cells. The protein kinase activity of immunoaffinity-purified ATR was dose-dependently decreased by SchB in vitro (IC₅₀: 7.25 μM), but the inhibitory effect was not observed in ATM, Chk1, PI3K, DNA-PK, and mTOR. The extent of UV-induced phosphorylation of p53 and Chk1 was markedly reduced by SchB in ATM-deficient but not siATR-treated cells. Taken together, our demonstration of the ability of SchB to inhibit ATR protein kinase activity following DNA damage in cells has clinical implications in anti-cancer therapy.     

Endothelin-1 stimulates catalase activity through the PKCδ-mediated phosphorylation of serine 167

Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and in lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H₂O₂) levels. These changes correlated with an increase in serine-phosphorylated catalase. Using the inhibitory peptide δV1.1, this phosphorylation was shown to be protein kinase Cδ (PKCδ) dependent. Mass spectrometry identified serine 167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from Escherichia coli or transiently transfected COS-7 cells demonstrated that S167D catalase had an increased ability to degrade H₂O₂ compared to the wild-type enzyme. Using a phospho-specific antibody, we were able to verify that pS167 catalase levels are modulated in lambs with acute increases in PBF in the presence and absence of the ET receptor antagonist tezosentan. S167 is located on the dimeric interface, suggesting it could be involved in regulating the formation of catalase tetramers. To evaluate this possibility we utilized analytical gel filtration to examine the multimeric structure of recombinant wild-type and S167D catalase. We found that recombinant wild-type catalase was present as a mixture of monomers and dimers, whereas S167D catalase was primarily tetrameric. Further, the incubation of wild-type catalase with PKCδ was sufficient to convert wild-type catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity.     

H2S contributed from CSE during cellular senescence suppresses inflammation and nitrosative stress

Aging involves the time-dependent deterioration of physiological functions attributed to various intracellular and extracellular factors. Cellular senescence is akin to aging and involves alteration in redox homeostasis. This is primarily marked by increased reactive oxygen/nitrogen species (ROS/RNS), inflammatory gene expression, and senescence-associated beta-galactosidase activity, all hallmarks of aging. It is proposed that gasotransmitters which include hydrogen sulfide (H₂S), carbon monoxide (CO), and nitric oxide (NO), may affect redox homeostasis during senescence. H₂S has been independently shown to induce DNA damage and suppress oxidative stress. While an increase in NO levels during aging is well established, the role of H₂S has remained controversial. To understand the role of H₂S during aging, we evaluated H₂S homeostasis in non-senescent and senescent cells, using a combination of direct measurements with a fluorescent reporter dye (WSP-5) and protein sulfhydration analysis. The free intracellular H₂S and total protein sulfhydration levels are high during senescence, concomitant to cystathionine gamma-lyase (CSE) expression induction. Using lentiviral shRNA-mediated expression knockdown, we identified that H₂S contributed by CSE alters global gene expression, which regulates key inflammatory processes during cellular senescence. We propose that H₂S decreases inflammation during cellular senescence by reducing phosphorylation of IκBα and the p65 subunit of nuclear factor kappa B (NF-κB). H₂S was also found to reduce NO levels, a significant source of nitrosative stress during cellular senescence. Overall, we establish H₂S as a key gasotransmitter molecule that regulates inflammatory phenotype and nitrosative stress during cellular senescence.     

Phosphorylation of cyclin D1 regulated by ATM or ATR controls cell cycle progression

Cyclin D1 is required at high levels for passage through G₁ phase but must be reduced to low levels during S phase to avoid the inhibition of DNA synthesis. This suppression requires the phosphorylation of Thr286, which is induced directly by DNA synthesis. Because the checkpoint kinase ATR is activated by normal replication as well as by DNA damage, its potential role in regulating cyclin D1 phosphorylation was tested. We found that ATR, activated by either UV irradiation or the topoisomerase IIβ binding protein 1 activator, promoted cyclin D1 phosphorylation. Small interfering RNA against ATR inhibited UV-induced Thr286 phosphorylation, together with that seen in normally cycling cells, indicating that ATR regulates cyclin D1 phosphorylation in normal as well as stressed cells. Following double-stranded DNA (dsDNA) breakage, the related checkpoint kinase ATM was also able to promote the phosphorylation of cyclin D1 Thr286. The relationship between these checkpoint kinases and cyclin D1 was extended when we found that normal cell cycle blockage in G₁ phase observed following dsDNA damage was efficiently overcome when exogenous cyclin D1 was expressed within the cells. These results indicate that checkpoint kinases play a critical role in regulating cell cycle progression in normal and stressed cells by directing the phosphorylation of cyclin D1.     

H2S protects from oxidative stress-driven ACE2 expression and cardiac aging

Cystathionine gamma-lyase (CSE)-derived hydrogen sulfide (H₂S) plays an essential role in preserving cardiac functions. Angiotensin-converting enzyme 2 (ACE2) acts as the negative regulator of the renin-angiotensin system, exerting anti-oxidative stress and anti-inflammatory properties within the body. The interplays of CSE/H₂S signaling and ACE2 in cardiac aging are unclear. In this study, the regulatory roles of H₂S on ACE2 expression in mouse heart tissue and rat cardiomyocytes under different stress conditions were investigated. It was found that ACE2 protein level was lower in heart tissues from old mice (56-week-old) than young mice (8-week-old), and the knockout of CSE (CSE KO) induced moderate oxidative stress and further inhibited ACE2 protein level in mouse hearts at both young and old age. Incubation of rat cardiac cells (H9C2) with a low dose of H₂O₂ (50 µM) suppressed ACE2 protein level and induced cellular senescence, which was completely reversed by co-incubation with 30 µM NaHS (a H₂S donor). Prolonged nutrient excess is an increased risk of heart disorders by causing metabolic dysfunction and cardiac remodeling. We further found high-fat diet feeding stimulated ACE2 expression and induced severe oxidative stress in CSE KO heart in comparison with wild-type heart. Lipid overload in H9C2 cells to mimic a status of nutrient excess also enhanced the expression of ACE2 protein and induced severe oxidative stress and cell senescence, which were significantly attenuated by the supplementation of exogenous H₂S. Furthermore, the manipulation of ACE2 expression partially abolished the protective role of H₂S against cellular senescence. These results demonstrate the dynamic roles of H₂S in the maintenance of ACE2 levels under different levels of oxidative stress, pointing to the potential implications in targeting the CSE/H₂S system for the interruption of aging and diabetes-related heart disorders.

     

Nek1 kinase functions in DNA damage response and checkpoint control through a pathway independent of ATM and ATR

Never-in-mitosis A related protein kinase 1 (Nek1) is involved early in a DNA damage sensing/repair pathway. We have previously shown that cells without functional Nek1 fail to activate the more distal kinases Chk1 and Chk2 and fail to arrest properly at G₁/S or M-phase checkpoints in response to DNA damage. As a consequence, foci of damaged DNA in Nek1 null cells persist long after the instigating insult, and Nek1 null cells develop unstable chromosomes at a rate much higher than identically cultured wild-type cells. Here we show that Nek1 functions independently of canonical DNA damage responses requiring the PI3 kinase-like proteins ATM and ATR. Chemical inhibitors of ATM/ATR or mutation of the genes that encode them fail to alter the kinase activity of Nek1 or its localization to nuclear foci of DNA damage. Moreover ATM and ATR activities, including the localization of the proteins to DNA damage sites and phosphorylation of early DNA damage response substrates, are intact in Nek1^−/− murine cells and in human cells with Nek1 expression silenced by siRNA. Our results demonstrate that Nek1 is important for proper checkpoint control and characterize for the first time a DNA damage response that does not directly involve one of the known upstream mediator kinases, ATM or ATR.Key words: checkpoint control, DNA damage response, Nek1, ATM, ATR     

Hydrogen Sulfide Suppresses Oxidized Low-density Lipoprotein (Ox-LDL)-stimulated Monocyte Chemoattractant Protein 1 generation from Macrophages via the Nuclear Factor κB (NF-κB) Pathway

This study was designed to examine the role of hydrogen sulfide (H₂S) in the generation of oxidized low-density lipoprotein (ox-LDL)-stimulated monocyte chemoattractant protein 1 (MCP-1) from macrophages and possible mechanisms. THP-1 cells and RAW macrophages were pretreated with sodium hydrosulfide (NaHS) and hexyl acrylate and then treated with ox-LDL. The results showed that ox-LDL treatment down-regulated the H₂S/cystathionine-β-synthase pathway, with increased MCP-1 protein and mRNA expression in both THP-1 cells and RAW macrophages. Hexyl acrylate promoted ox-LDL-induced inflammation, whereas the H₂S donor NaHS inhibited it. NaHS markedly suppressed NF-κB p65 phosphorylation, nuclear translocation, DNA binding activity, and recruitment to the MCP-1 promoter in ox-LDL-treated macrophages. Furthermore, NaHS decreased the ratio of free thiol groups in p65, whereas the thiol reductant DTT reversed the inhibiting effect of H₂S on the p65 DNA binding activity. Most importantly, site-specific mutation of cysteine 38 to serine in p65 abolished the effect of H₂S on the sulfhydration of NF-κB and ox-LDL-induced NF-κB activation. These results suggested that endogenous H₂S inhibited ox-LDL-induced macrophage inflammation by suppressing NF-κB p65 phosphorylation, nuclear translocation, DNA binding activity, and recruitment to the MCP-1 promoter. The sulfhydration of free thiol group on cysteine 38 in p65 served as a molecular mechanism by which H₂S inhibited NF-κB pathway activation in ox-LDL-induced macrophage inflammation.     

Serine 1179 Phosphorylation of Endothelial Nitric Oxide Synthase Increases Superoxide Generation and Alters Cofactor Regulation

Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. In addition to producing NO, eNOS can also generate superoxide (O₂ ^-.) in the absence of the cofactor tetrahydrobiopterin (BH4). Previous studies have shown that bovine eNOS serine 1179 (Serine 1177/human) phosphorylation critically modulates NO synthesis. However, the effect of serine 1179 phosphorylation on eNOS superoxide generation is unknown. Here, we used the phosphomimetic form of eNOS (S1179D) to determine the effect of S1179 phosphorylation on superoxide generating activity, and its sensitivity to regulation by BH4, Ca²⁺, and calmodulin (CAM). S1179D eNOS exhibited significantly increased superoxide generating activity and NADPH consumption compared to wild-type eNOS (WT eNOS). The superoxide generating activities of S1179D eNOS and WT eNOS did not differ significantly in their sensitivity to regulation by either Ca²⁺ or CaM. The sensitivity of the superoxide generating activity of S1179D eNOS to inhibition by BH4 was significantly reduced compared to WT eNOS. In eNOS-overexpressing 293 cells, BH4 depletion with 10mM DAHP for 48 hours followed by 50ng/ml VEGF for 30 min to phosphorylate eNOS S1179 increased ROS accumulation compared to DAHP-only treated cells. Meanwhile, MTT assay indicated that overexpression of eNOS in HEK293 cells decreased cellular viability compared to control cells at BH4 depletion condition (P<0.01). VEGF-mediated Serine 1179 phosphorylation further decreased the cellular viability in eNOS-overexpressing 293 cells (P<0.01). Our data demonstrate that eNOS serine 1179 phosphorylation, in addition to enhancing NO production, also profoundly affects superoxide generation: S1179 phosphorylation increases superoxide production while decreasing sensitivity to the inhibitory effect of BH4 on this activity.     

Inhibition of cellular proliferation by the genistein metabolite 5,7,3',4'-tetrahydroxyisoflavone is mediated by DNA damage and activation of the ATR signalling pathway

The cellular actions of genistein, and its in vivo metabolites, are believed to mediate the decreased risk of breast cancer associated with high soy consumption. The genistein metabolite, 5,7,3′,4′-tetrahydroxyisoflavone (THIF), induced G2-M cell cycle arrest in T47D tumorigenic breast epithelial cells via a mechanism involving the activation of ataxia telangiectasia and Rad3-related kinase (ATR) via its phosphorylation at Ser⁴²⁸. This activation of ATR appeared to result from THIF-induced increases in intracellular oxidative stress, a depletion of cellular GSH and an increase in DNA strand breakage. THIF treatment also led to an inhibition of cdc2, which was accompanied by the phosphorylation of both p53 (Ser¹⁵) and Chk1 (Ser²⁹⁶) and the de-activation of cdc25C phosphatase. We suggest the anti-proliferative actions of THIF may be mediated by initial oxidative DNA damage, activation of ATR and downstream regulation of the p53 and Chk1 pathways leading to cell cycle arrest in G2-M. This may represent one mechanism by which genistein exerts its cellular activity in vivo.     

Neuroprotectin D1 Induces Dephosphorylation of Bcl-xL in a PP2A-dependent Manner during Oxidative Stress and Promotes Retinal Pigment Epithelial Cell Survival

Retinal pigment epithelial (RPE) cell integrity is critical for the survival of photoreceptor cells. Bcl-x_L is a major anti-apoptotic Bcl-2 protein required for RPE cell survival, and phosphorylation of Bcl-x_L at residue Ser-62 renders this protein pro-apoptotic. In this study, we identify serine/threonine protein phosphatase 2A (PP2A) as a key regulator of Bcl-x_L phosphorylation at residue Ser-62 in ARPE-19 cells, a spontaneously arising RPE cell line in which Bcl-x_L is highly expressed. We found that either PP2A inhibitor okadaic acid or depletion of catalytic subunit α of PP2A (PP2A/Cα) by small interfering RNA enhanced Bcl-x_L phosphorylation when activated with hydrogen peroxide and tumor necrosis factor α-induced oxidative stress. Disruption of PP2A/Cα exacerbated oxidative stress-induced apoptosis. PP2A/Cα colocalized and interacted with S62Bcl-x_L in cells stressed with H₂O₂/tumor necrosis factor α. By contrast, the omega-3 fatty acid docosahexaenoic acid derivative, neuroprotectin D1 (NPD1), a potent activator of survival signaling, down-regulated oxidative stress-induced phosphorylation of Bcl-x_L by increasing protein phosphatase activity. NPD1 also attenuated the oxidative stress-induced apoptosis by knockdown of PP2A/Cα and increased the association of PP2A/Cα with S62Bcl-x_L as well as total Bcl-x_L. NPD1 also enhanced the heterodimerization of Bcl-x_L with its counterpart, pro-apoptotic protein Bax. Thus, NPD1 modulates the activation of this Bcl-2 family protein by dephosphorylating in a PP2A-dependent manner, suggesting a coordinated, NPD1-mediated regulation of cell survival in response to oxidative stress.     

Identification and characterization of PP2C phosphatase SjPtc1 in Schistosoma japonicum

Protein phosphorylation, regulated by protein kinases and protein phosphatases, is crucial for protein structure and function in eukaryotic organisms. Type 2C protein phosphatase (PP2C) belongs to the serine/threonine phosphatase family and its activities require the presence of a divalent magnesium or manganese ion. In the present study, a potential PP2C phosphatase (SjPtc1) was identified in Schistosoma japonicum. The SjPTC1 gene was found to be highly expressed in adult worms. A recombinant SjPtc1 protein showed typical PP2C phosphatase activity. Heterologous SjPTC1 expression reversed the sensitivity of yeast ptc1 null mutants toward H₂O₂, ZnCl₂, cisplatin, and rapamycin. Collectively, the results suggest that SjPtc1 may take part in the regulation of cellular responses to oxidative stress, DNA damage stress, and the TOR (target of rapamycin) signaling pathway.     

S‐sulfhydration of MEK1 leads to PARP‐1 activation and DNA damage repair

The repair of DNA damage is fundamental to normal cell development and replication. Hydrogen sulfide (H₂S) is a novel gasotransmitter that has been reported to protect cellular aging. Here, we show that H₂S attenuates DNA damage in human endothelial cells and fibroblasts by S‐sulfhydrating MEK1 at cysteine 341, which leads to PARP‐1 activation. H₂S‐induced MEK1 S‐sulfhydration facilitates the translocation of phosphorylated ERK1/2 into nucleus, where it activates PARP‐1 through direct interaction. Mutation of MEK1 cysteine 341 inhibits ERK phosphorylation and PARP‐1 activation. In the presence of H₂S, activated PARP‐1 recruits XRCC1 and DNA ligase III to DNA breaks to mediate DNA damage repair, and cells are protected from senescence.     

ATR is not required for p53 activation but synergizes with p53 in the replication checkpoint.

ATR (ataxia telangiectasia and Rad-3-related) is a protein kinase required for survival after DNA damage. A critical role for ATR has been hypothesized to be the regulation of p53 and other cell cycle checkpoints. ATR has been shown to phosphorylate p53 at Ser(15), and this damage-induced phosphorylation is diminished by expression of a catalytically inactive (ATR-kd) mutant. p53 function could not be examined directly in prior studies of ATR, however, because p53 was mutant or because cells expressed the SV40 large T antigen that blocks p53 function. To test the interactions of ATR and p53 directly we generated human U2OS cell lines inducible for either wild-type or kinase-dead ATR that also have an intact p53 pathway. Indeed, ATR-kd expression sensitized these cells to DNA damage and caused a transient decrease in damage-induced serine 15 phosphorylation of p53. However, we found that the effects of ATR-kd expression do not result in blocking the response of p53 to DNA damage. Specifically, prior ATR-kd expression had no effect on DNA damage-induced p53 protein up-regulation, p53-DNA binding, p21 mRNA up-regulation, or G(1) arrest. Instead of promoting survival via p53 regulation, we found that ATR protects cells by delaying the generation of mitotic phosphoproteins and inhibiting premature chromatin condensation after DNA damage or hydroxyurea. Although p53 inhibition (by E6 or MDM2 expression) had little effect on premature chromatin condensation, when combined with ATR-kd expression there was a marked loss of the replication checkpoint. We conclude that ATR and p53 can function independently but that loss of both leads to synergistic disruption of the replication checkpoint.     

Oxidative Stress-Induced DNA Damage and Repair in Human Peripheral Blood Mononuclear Cells: Protective Role of Hemoglobin

Background

DNA repair is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. There is a controversy with regard to the effect of red blood cells on DNA damage and cellular response.

Aim

To investigate the effect of red blood cells on H₂O₂-induced DNA damage and repair in human peripheral blood mononuclear cells.

Methods

DNA breaks were induced in peripheral blood mononuclear cells by H₂O₂ in the absence or presence of red blood cells, red blood cells hemolysate or hemoglobin. DNA repair was measured by ³H-thymidine uptake, % double-stranded DNA was measured by fluorometric assay of DNA unwinding. DNA damage was measured by the comet assay and by the detection of histone H2AX phosphorylation.

Results

Red blood cells and red blood cells hemolysate reduced DNA repair in a dose-dependent manner. Red blood cells hemolysate reduced % double-stranded DNA, DNA damage and phosphorylation of histone H2AX. Hemoglobin had the same effect as red blood cells hemolysate on % double-stranded DNA.

Conclusion

Red blood cells, via red blood cells hemolysate and hemoglobin, reduced the effect of oxidative stress on peripheral blood mononuclear cell DNA damage and phosphorylation of histone H2AX. Consequently, recruitment of DNA repair proteins diminished with reduction of DNA repair. This suggests that anemia predisposes to increased oxidative stress induced DNA damage, while a higher hemoglobin level provides protection against oxidative-stress-induced DNA damage.     

Hydrogen peroxide impairs insulin-stimulated assembly of mTORC1

Oxidants are well recognized for their capacity to reduce the phosphorylation of the mammalian target of rapamycin (mTOR) substrates, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and p70 S6 kinase 1 (S6K1), thereby hindering mRNA translation at the level of initiation. mTOR functions to regulate mRNA translation by forming the signaling complex mTORC1 (mTOR, raptor, GβL). Insulin signaling to mTORC1 is dependent upon phosphorylation of Akt/PKB and the inhibition of the tuberous sclerosis complex (TSC1/2), thereby enhancing the phosphorylation of 4E-BP1 and S6K1. In this study we report the effect of H₂O₂ on insulin-stimulated mTORC1 activity and assembly using A549 and bovine aortic smooth muscle cells. We show that insulin stimulated the phosphorylation of TSC2 leading to a reduction in raptor–mTOR binding and in the quantity of proline-rich Akt substrate 40 (PRAS40) precipitating with mTOR. Insulin also increased 4E-BP1 coprecipitating with mTOR and the phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1. H₂O₂, on the other hand, opposed the effects of insulin by increasing raptor–mTOR binding and the ratio of PRAS40/raptor derived from the mTOR immunoprecipitates in both cell types. These effects occurred in conjunction with a reduction in 4E-BP1 phosphorylation and the 4E-BP1/raptor ratio. siRNA-mediated knockdown of PRAS40 in A549 cells partially reversed the effect of H₂O₂ on 4E-BP1 phosphorylation but not on S6K1. These findings are consistent with PRAS40 functioning as a negative regulator of insulin-stimulated mTORC1 activity during oxidant stress.     

Activation of DNA damage response pathways in human mesenchymal stem cells exposed to cisplatin or γ-irradiation

DNA damaging agents are widely used in treatment of hematogical malignancies and solid tumors. While effects on hematopoietic stem cells have been characterized, less is known about the DNA damage response in human mesenchymal stem cells (hMSCs) in the bone marrow stroma, progenitors of osteoblasts, chondrocytes and adipocytes. To elucidate the response of undifferentiated hMSCs to γ-irradiation and cisplatin, key DNA damage responses have been characterised in hMSCs from normal adult donors. Cisplatin and γ-irradiation activated the DNA damage response in hMSCs, including induction of p53 and p21, and activation of PI3 kinase-related protein kinase (PIKK)-dependent phosphorylation of histone H2AX on serine 139, and replication protein A2 on serine4/serine8. Chemical inhibition of ATM or DNA-PK reduced DNA damage-induced phosphorylation of H2AX, indicating a role for both PIKKs in the response of hMSCs to DNA damage. Consistent with repair of DNA strand breaks, γ-H2AX staining decreased by 24 hours following gamma-irradiation. γ-irradiation arrested hMSCs in the G₁ phase of the cell cycle, while cisplatin induced S-phase arrest, mediated in part by the ATR/Chk1 checkpoint pathway. In hMSCs isolated from a chronic lymphocytic leukemia (CLL) patient, p53 and p21 were induced by cisplatin and γ-irradiation, while RPA2 was phosphorylated on serine4/8 in particular following cisplatin. Compared to peripheral blood lymphocytes or the leukemia cell line K562, both normal hMSCs and CLL-derived hMSCs were more resistant to cisplatin and γ-irradiation. These results provide insights into key pathways mediating the response of bone marrow-derived hMSCs to DNA damaging agents used in cancer treatment.     

Inhibition of Syk promotes chemical reprogramming of fibroblasts via metabolic rewiring and H2S production

Chemical compounds have recently been introduced as alternative and non‐integrating inducers of pluripotent stem cell fate. However, chemical reprogramming is hampered by low efficiency and the molecular mechanisms remain poorly characterized. Here, we show that inhibition of spleen tyrosine kinase (Syk) by R406 significantly promotes mouse chemical reprogramming. Mechanistically, R406 alleviates Syk / calcineurin (Cn) / nuclear factor of activated T cells (NFAT) signaling‐mediated suppression of glycine, serine, and threonine metabolic genes and dependent metabolites. Syk inhibition upregulates glycine level and downstream transsulfuration cysteine biosynthesis, promoting cysteine metabolism and cellular hydrogen sulfide (H₂S) production. This metabolic rewiring decreased oxidative phosphorylation and ROS levels, enhancing chemical reprogramming. In sum, our study identifies Syk‐Cn‐NFAT signaling axis as a new barrier of chemical reprogramming and suggests metabolic rewiring and redox homeostasis as important opportunities for controlling cell fates.     

Crosstalk between JNK and SUMO signaling pathways: deSUMOylation is protective against H2O2-induced cell injury

Background

Oxidative stress is a key feature in the pathogenesis of several neurological disorders. Following oxidative stress stimuli a wide range of pathways are activated and contribute to cellular death. The mechanism that couples c-Jun N-terminal kinase (JNK) signaling, a key pathway in stress conditions, to the small ubiquitin-related modifier (SUMO), an emerging protein in the field, is largely unknown.

Methodology/Principal Findings

With this study we investigated if SUMOylation participates in the regulation of JNK activation as well as cellular death in a model of H₂O₂ induced-oxidative stress. Our data show that H₂O₂ modulates JNK activation and induces cellular death in neuroblastoma SH-SY5Y cells. Inhibition of JNK''s action with the D-JNKI1 peptide rescued cells from death. Following H₂O₂, SUMO-1 over-expression increased phosphorylation of JNK and exacerbated cell death, although only in conditions of mild oxidative stress. Furthermore inhibition of SUMOylation, following transfection with SENP1, interfered with JNK activation and rescued cells from H₂O₂ induced death. Importantly, in our model, direct interaction between these proteins can occur.

Conclusions/Significance

Taken together our results show that SUMOylation may significantly contribute to modulation of JNK activation and contribute to cell death in oxidative stress conditions.