Regulation of NF-κB signalling by the mono-ADP-ribosyltransferase ARTD10.

Adenosine diphosphate-ribosylation is a post-translational modification mediated by intracellular and membrane-associated extracellular enzymes and many bacterial toxins. The intracellular enzymes modify their substrates either by poly-ADP-ribosylation, exemplified by ARTD1/PARP1, or by mono-ADP-ribosylation. The latter has been discovered only recently, and little is known about its physiological relevance. The founding member of mono-ADP-ribosyltransferases is ARTD10/PARP10. It possesses two ubiquitin-interaction motifs, a unique feature among ARTD/PARP enzymes. Here, we find that the ARTD10 ubiquitin-interaction motifs bind to K63-linked poly-ubiquitin, a modification that is essential for NF-κB signalling. We therefore studied the role of ARTD10 in this pathway. ARTD10 inhibits the activation of NF-κB and downstream target genes in response to interleukin-1ß and tumour necrosis factor-α, dependent on catalytic activity and poly-ubiquitin binding of ARTD10. Mechanistically ARTD10 interferes with poly-ubiquitination of NEMO, which interacts with and is a substrate of ARTD10. Our findings identify a novel regulator of NF-κB signalling and provide evidence for cross-talk between K63-linked poly-ubiquitination and mono-ADP-ribosylation.

Depletion of tristetraprolin in breast cancer cells increases interleukin-16 expression and promotes tumor infiltration with monocytes/macrophages.

The RNA-binding protein tristetraprolin (TTP) destabilizes target messenger RNAs (mRNAs) containing AU-rich elements within their 3' untranslated region. Thereby, it controls the expression of multiple inflammatory and tumor-associated transcripts. Moreover, a loss of TTP in tumors predicts disease-associated survival. Although tumor intrinsic functions of TTP have previously been studied, the impact of TTP on the interaction of tumors with their microenvironment remains elusive. As immune cell infiltration into tumors is a critical determinant for tumor progression, this study aimed at determining the influence of tumor cell TTP on the interaction between tumor and immune cells, specifically monocytes (MO)/macrophages (MΦ). Knockdown (k/d) of TTP in T47D breast cancer cells enhanced tumor growth both in vitro and in vivo and increased infiltration of MO into 3D tumor spheroids in vitro and of MΦ into tumor xenografts in vivo. Enhanced migration of MO toward supernatants of TTP-deficient tumor spheroids was determined as the underlying principle. Interestingly, we noticed interleukin-16 (IL-16) mRNA stabilization when TTP was depleted. In line, IL-16 protein levels were elevated in TTP-deficient spheroids and their supernatants as well as in TTP k/d tumor xenografts and critically contributed to the enhanced chemotactic behavior. In summary, we show that the loss of TTP in tumors not only affects tumor cell proliferation and survival but also enhances infiltration of MO/MΦ into the tumors, which is typically associated with poor prognosis. Moreover, we identified IL-16 as a critical TTP-regulated chemotactic factor that contributes to MO/MΦ migration.

Erioflorin stabilizes the tumor suppressor Pdcd4 by inhibiting its interaction with the E3-ligase ß-TrCP1.

Loss of the tumor suppressor Pdcd4 was reported for various tumor entities and proposed as a prognostic marker in tumorigenesis. We previously characterized decreased Pdcd4 protein stability in response to mitogenic stimuli, which resulted from p70(S6K1)-dependent protein phosphorylation, ß-TrCP1-mediated ubiquitination, and proteasomal destruction. Following high-throughput screening of natural product extract libraries using a luciferase-based reporter assay to monitor phosphorylation-dependent proteasomal degradation of the tumor suppressor Pdcd4, we succeeded in showing that a crude extract from Eriophyllum lanatum stabilized Pdcd4 from TPA-induced degradation. Erioflorin was identified as the active component and inhibited not only degradation of the Pdcd4-luciferase-based reporter but also of endogenous Pdcd4 at low micromolar concentrations. Mechanistically, erioflorin interfered with the interaction between the E3-ubiquitin ligase ß-TrCP1 and Pdcd4 in cell culture and in in vitro binding assays, consequently decreasing ubiquitination and degradation of Pdcd4. Interestingly, while erioflorin stabilized additional ß-TrCP-targets (such as IκBα and ß-catenin), it did not prevent the degradation of targets of other E3-ubiquitin ligases such as p21 (a Skp2-target) and HIF-1α (a pVHL-target), implying selectivity for ß-TrCP. Moreover, erioflorin inhibited the tumor-associated activity of known Pdcd4- and IκBα-regulated αtranscription factors, that is, AP-1 and NF-κB, altered cell cycle progression and suppressed proliferation of various cancer cell lines. Our studies succeeded in identifying erioflorin as a novel Pdcd4 stabilizer that inhibits the interaction of Pdcd4 with the E3-ubiquitin ligase ß-TrCP1. Inhibition of E3-ligase/target-protein interactions may offer the possibility to target degradation of specific proteins only as compared to general proteasome inhibition.

HIF-1α protein is upregulated in HIF-2α depleted cells via enhanced translation.

The hypoxia-inducible factors HIF-1 and HIF-2 are primarily regulated via stabilization of their respective ?-subunits under hypoxic conditions. Previously, compensatory upregulation of one HIF-α-subunit upon depletion of the other α-subunit was described, yet the underlying mechanism remained elusive. Here we provide evidence that enhanced HIF-1α protein expression in HIF-2α knockdown (k/d) cells neither results from elevated HIF-1α mRNA expression, nor from increased HIF-1α protein stability. Instead, we identify enhanced HIF-1α translation as molecular mechanism. Moreover, we found elevated levels of the RNA-binding protein HuR and provide evidence that HuR is critical for the compensatory HIF-1α regulation in HIF-2α k/d cells.

Inflammation-induced loss of Pdcd4 is mediated by phosphorylation-dependent degradation.

The tumor suppressor programmed cell death 4 (Pdcd4) is lost in various tumor tissues. Loss of Pdcd4 has been associated with increased tumorigenic potential and tumor progression. While various mechanisms of Pdcd4 regulation have been described, the effect of an inflammatory tumor microenvironment on Pdcd4 protein expression has not been characterized so far. In the present study, we aimed to elucidate the molecular mechanisms of Pdcd4 protein regulation in tumor cells under inflammatory conditions. 12-O-tetradecanoylphorbol 13-acetate-induced differentiation of human U937 monocytes increased the expression and secretion of inflammatory cytokines such as tumor necrosis factor α, interleukin (IL)-6 and IL-8. Exposure to conditioned medium (CM) of these activated macrophages markedly decreased Pdcd4 protein expression in various tumor cells. Similarly, indirect coculture with such activated U937 monocyte-derived macrophages resulted in the loss of Pdcd4 protein in tumor cells. Decreased Pdcd4 protein levels were attributable to enhanced proteasomal degradation, diminishing Pdcd4 protein half-life. Proteasomal degradation required activation of phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signaling. Since macrophage-CM sufficed to induce Pdcd4 degradation, Pdcd4 downregulation was determined to be an indirect unidirectional effect of the macrophages on the tumor cells. Pdcd4 protein expression was also attenuated in vivo in mouse colon tissue in response to dextran sodium sulfate-induced colitis. In summary, we characterized PI3K-mTOR-dependent proteasome-mediated Pdcd4 degradation in tumor cells in the inflammatory tumor microenvironment. Consequently, stabilization of Pdcd4 protein could provide a promising novel avenue for therapeutics targeting inflammation-associated tumors.

A combination of hypoxia and lipopolysaccharide activates tristetraprolin to destabilize proinflammatory mRNAs such as tumor necrosis factor-alpha.

Inflammation is often accompanied by hypoxia because of the high oxygen consumption of invading bacteria and immune cells. During resolution of inflammation, the formation of inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha), which is produced by macrophages, needs to be terminated. We show in RAW264.7 macrophages that TNF-alpha mRNA as well as intracellular and secreted TNF-alpha protein levels are reduced after prolonged incubations with lipopolysaccharide (LPS) under hypoxic conditions. The decrease in TNF-alpha was mediated by destabilization of TNF-alpha mRNA via a 3'-untranslated region-dependent mechanism. Specifically, the RNA-binding protein tristetraprolin (TTP) increased at mRNA and protein levels after 16-hour incubations with LPS under hypoxia. Interestingly, TTP accumulated in a dephosphorylated and active form, and this accumulation was attributable to reduced p38 mitogen-activated protein kinase activity under these conditions. Knockdown of TTP by small interfering RNA abolished destabilization of TNF-alpha mRNA. Prolonged incubations with LPS under hypoxia also reduced mRNA amounts and stability of other proinflammatory mediators such as macrophage inflammatory protein-2, interleukin-6, and granulocyte macrophage colony-stimulating factor. Therefore, we propose that hypoxia plays a key role during resolution of inflammation by activating posttranscriptional, TTP-dependent regulatory mechanisms.