Interferon-␥ (IFN␥) is a pluripotent cytokine whose major biological effects are mediated through a pathway in which STAT1 is the predominant and essential transcription factor. STAT3 can also be activated weakly by IFN␥, but the mechanism of activation and function of STAT3 as a part of the interferon response are not known. Here we show that STAT3 activation is much stronger and more prolonged in STAT1-null mouse embryo fibroblasts than in wild-type cells. In response to IFN␥, SRC-family kinases are required to activate STAT3 (but not STAT1) through tyrosine phosphorylation, whereas the receptor-bound kinases JAK1 and JAK2 are required to activate both STATs.
Nonischemic cardiomyopathy (NICM) resulting from long-standing hypertension, valvular disease, and genetic mutations is a major cause of heart failure worldwide. Recent observations suggest that myeloid cells can impact cardiac function, but the role of tissue-intrinsic vs. tissue-extrinsic myeloid cells in NICM remains poorly understood. Here, we show that cardiac resident macrophage proliferation occurs within the first week following pressure overload hypertrophy (POH; a model of heart failure) and is requisite for the heart's adaptive response. Mechanistically, we identify Kruppel-like factor 4 (KLF4) as a key transcription factor that regulates cardiac resident macrophage proliferation and angiogenic activities. Finally, we show that blood-borne macrophages recruited in late-phase POH are detrimental, and that blockade of their infiltration improves myocardial angiogenesis and preserves cardiac function. These observations demonstrate previously unappreciated temporal and spatial roles for resident and nonresident macrophages in the development of heart failure.
Suppressor of cytokine signaling (SOCS)-1, the key negative regulator of interferon (IFN)-␥-dependent signaling, is induced in response to IFN␥. SOCS-1 binds to and inhibits the IFN␥ receptor-associated kinase Janusactivated kinase (JAK) 2 and inhibits its function in vitro, but the mechanism by which SOCS-1 inhibits IFN␥-dependent signaling in vivo is not clear. Upon stimulation, mouse IFN␥ receptor subunit 1 (IFNGR1) is phosphorylated on several cytoplasmic tyrosine residues, and Tyr 419 is required for signal transducer and activator of transcription (STAT) 1 activation in mouse embryo fibroblasts. However, the functions of the other three cytoplasmic tyrosine residues are not known. Here we show that Tyr 441 is required to attenuate STAT1 activation in response to IFN␥. Several tyrosine to phenylalanine mutants of IFNGR1, expressed at normal levels in stable pools of IFNGR1-null cells, were analyzed for the phosphorylation of STAT1 during a 48-h period, and antiviral activity in response to IFN␥ was also measured. Stronger activation of STAT1 was observed in cells expressing all IFNGR1 variants mutated at Tyr 441 , and, consistently, stronger antiviral activity was also observed in these cells. Interferon (IFN)1 -␥ plays key roles in mediating antiviral and antigrowth responses and in modulating immune responses (1). The major signal transduction pathway activated by IFN␥ has been elucidated through both biochemical and genetic studies. The IFN␥ receptor complex consists of two receptor subunits, IFNGR1 and IFNGR2, and the tyrosine kinases Janus-activated kinase (JAK) 1 and JAK2, which bind to IFNGR1 and IFNGR2, respectively. IFN␥ induces the oligomerization of the receptor subunits, leading to the activation of JAK1 and JAK2, which then phosphorylate tyrosine residues within the cytoplasmic domain of IFNGR1. Signal transducer and activator of transcription (STAT) 1 is then recruited to the receptor complex and phosphorylated on Tyr 701 , allowing it to be released, form homodimers, translocate to the nucleus, and bind to ␥-activated sequences to activate the transcription of interferon-stimulated genes (ISGs) (1-3).The activation of STAT1 by IFN␥ is tightly controlled by several mechanisms (4). The SH2-containing phosphatase 2 binds to IFNGR1 and inhibits STAT1 activation without inhibiting the phosphorylation of IFNGR1 (5). Protein inhibitor of activated STAT 1 (PIAS-1) binds to STAT1 and prevents its association with target DNA (6). Both genetic and biochemical studies have shown that suppressor of cytokine signaling (SOCS)-1 is the most potent inhibitor of IFN␥ signaling (7). Mice lacking SOCS-1 develop a complex fatal neonatal disease (8 -10), and the mortality, which results from hypersensitivity to IFN␥, is largely prevented by administration of anti-IFN␥. In addition, premature death does not occur in mice lacking both 11). In response to IFN␥, STAT1 activation is much stronger in cells lacking SOCS-1 than in wild-type cells (11,12). The constitutive expression of SOCS-1 blocks IFN␥-mediated antivir...
To every Gromov hyperbolic space X one can associate a space at infinity called the Gromov boundary of X. Gromov showed that quasi-isometries of hyperbolic metric spaces induce homeomorphisms on their boundaries, thus giving rise to a well-defined notion of the boundary of a hyperbolic group. Croke and Kleiner showed that the visual boundary of non-positively curved (CAT(0)) groups is not well-defined, since quasi-isometric CAT(0) spaces can have non-homeomorphic boundaries. For any sublinear function κ, we consider a subset of the visual boundary called the κ-Morse boundary and show that it is QI-invariant and metrizable. This is to say, the κ-Morse boundary of a CAT(0) group is well-defined. In the case of Right-angled Artin groups, it is shown in the Appendix that the Poisson boundary of random walks is naturally identified with the ( √ t log t)-boundary.
A r t i c l e A m e n d m e n t s2At the request of the corresponding author, John W. Hollingsworth, and the last author, David A. Schwartz, the JCI is retracting this paper.Following an inquiry at Duke University, Drs. Hollingsworth and Schwartz were informed that the flexiVent data depicted in Figure 1A and Supplemental Figure 1A provided by the animal pulmonary physiology laboratory at Duke University may have been unreliable. Dr. Schwartz therefore repeated the experiments shown in Figure 1A using different flexiVent equipment with a limited number of experimental animals and was unable to replicate the airway hyperresponsiveness findings. The authors have stated that the other findings presented in the article were generated and analyzed in Dr. Schwartz's laboratory and are not affected by the unreliable flexiVent data produced by the animal pulmonary physiology laboratory at Duke University. In the left panel of Figure 8A, the P value for comparing KO Ang II with KO Ang II-Cl10400 was incorrect in the print version and the original online version of this article; a correct version of the latter has since been published. The correct figure panel is below. ErratumThe JCI regrets the error.
5-fluorodeoxyuridine (5-FdU, floxuridine) is active against multiple cancers through the inhibition of thymidylate synthase, which consequently introduces uracil and 5-FU incorporation into the genome. Uracil DNA glycosylase (UDG) is one of the main enzymes responsible for the removal of uracil and 5-FU. However, how exactly UDG mediates cellular sensitivity to 5-FdU, and if so whether it is through its ability to remove uracil and 5-FU have not been well characterized. In this study, we report that UDG depletion led to incorporation of uracil and 5-FU in DNA following 5-FdU treatment and significantly enhanced 5-FdU's cytotoxicity in cancer cell lines. Co-treatment, but not post-treatment with thymidine prevented cell death of UDG depleted cells by 5-FdU, indicating that the enhanced cytotoxicity is due to the retention of uracil and 5-FU in genomic DNA in the absence of UDG. Furthermore, UDG depleted cells were arrested at late G1 and early S phase by 5-FdU, followed by accumulation of sub-G1 population indicating cell death. Mechanistically, 5-FdU dramatically reduced DNA replication speed in UDG depleted cells. UDG depletion also greatly enhanced DNA damage as shown by γH2AX foci formation. Notably, the increased γH2AX foci formation was not suppressed by caspase inhibitor treatment, suggesting that DNA damage precedes cell death induced by 5-FdU. Together, these data provide novel mechanistic insights into the roles of UDG in DNA replication, damage repair, and cell death in response to 5-FdU and suggest that UDG is a target for improving the anticancer effect of this agent.
Thymidylate synthase (TS) inhibitors including fluoropyrimidines [e.g., 5-Fluorouracil (5-FU) and 5-Fluorodeoxyuridine (5-FdU, floxuridine)] and antifolates (e.g., pemetrexed) are widely used against solid tumors. Previously, we reported that shRNA-mediated knockdown (KD) of uracil DNA glycosylase (UDG) sensitized cancer cells to 5-FdU. Because p53 has also been shown as a critical determinant of the sensitivity to TS inhibitors, we further interrogated 5-FdU cytotoxicity after UDG depletion with regard to p53 status. By analyzing a panel of human cancer cells with known p53 status, it was determined that p53-mutated or -deficient cells are highly resistant to 5-FdU. UDG depletion resensitizes 5-FdU in p53-mutant and -deficient cells, whereas p53 wild-type (WT) cells are not affected under similar conditions. Utilizing paired HCT116 p53 WT and p53 knockout (KO) cells, it was shown that loss of p53 improves cell survival after 5-FdU, and UDG depletion only significantly sensitizes p53 KO cells. This sensitization can also be recapitulated by UDG depletion in cells with p53 KD by shRNAs. In addition, sensitization is also observed with pemetrexed in p53 KO cells, but not with 5-FU, most likely due to RNA incorporation. Importantly, in p53 WT cells, the apoptosis pathway induced by 5-FdU is activated independent of UDG status. However, in p53 KO cells, apoptosis is compromised in UDG-expressing cells, but dramatically elevated in UDG-depleted cells. Collectively, these results provide evidence that loss of UDG catalyzes significant cell death signals only in cancer cells mutant or deficient in p53. This study reveals that UDG depletion restores sensitivity to TS inhibitors and has chemotherapeutic potential in the context of mutant or deficient p53. .
Interleukin-1 (IL-1) induces the phosphorylation of Stat1 on serine 727 but not on tyrosine 701. Analyses of mutant I1A cells, which lack the IL-1 receptor-associated kinase (IRAK), and of I1A cells reconstituted with deletion mutants of IRAK show that the IL-1-mediated phosphorylation of Stat1 on serine requires the IRAK protein but not its kinase activity and does not involve phosphatidylinositol-3'-kinase (PI3K) or the mitogen-activated protein (MAP) kinases p38 or ERK. IRAK and Stat1 interact in vivo, and this interaction is increased in response to IL-1, suggesting that IRAK may serve to recruit the as yet unknown IL-1-induced Stat1 serine kinase. Chemical inhibitors or dominant-negative forms of signaling components required to activate NF-kappa B, ATF, or AP-1 in response to IL-1 do not affect the phosphorylation of Stat1 on serine. IL-1 and tumor necrosis factor (TNF) enhance the serine phosphorylation of Stat1 that occurs in response to interferon-gamma (IFN-gamma) and potentiate IFN-gamma-mediated, Stat1-driven gene expression, thus contributing to the synergistic activities of these proinflammatory cytokines.
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