Tobias Schmid

Redox Control of Inflammation in Macrophages

Macrophages are present throughout the human body, constitute important immune effector cells, and have variable roles in a great number of pathological, but also physiological, settings. It is apparent that macrophages need to adjust their activation profile toward a steadily changing environment that requires altering their phenotype, a process known as macrophage polarization. Formation of reactive oxygen species (ROS), derived from NADPH-oxidases, mitochondria, or NO-producing enzymes, are not necessarily toxic, but rather compose a network signaling system, known as redox regulation. Formation of redox signals in classically versus alternatively activated macrophages, their action and interaction at the level of key targets, and the resulting physiology still are insufficiently understood. We review the identity, source, and biological activities of ROS produced during macrophage activation, and discuss how they shape the key transcriptional responses evoked by hypoxia-inducible transcription factors, nuclear-erythroid 2-p45-related factor 2 (Nrf2), and peroxisome proliferator-activated receptor-γ. We summarize the mechanisms how redox signals add to the process of macrophage polarization and reprogramming, how this is controlled by the interaction of macrophages with their environment, and addresses the outcome of the polarization process in health and disease. Future studies need to tackle the option whether we can use the knowledge of redox biology in macrophages to shape their mediator profile in pathophysiology, to accelerate healing in injured tissue, to fight the invading pathogens, or to eliminate settings of altered self in tumors.

MicroRNA-27b Contributes to Lipopolysaccharide-mediated Peroxisome Proliferator-activated Receptor γ (PPARγ) mRNA Destabilization

Peroxisome proliferator-activated receptor γ (PPARγ) gained considerable interest as a therapeutic target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer is associated with impaired PPARγ expression. Considering that regulation of PPARγ expression during inflammation is largely unknown, we were interested in elucidating underlying mechanisms. To this end, we initiated an inflammatory response by exposing primary human macrophages to lipopolysaccharide (LPS) and observed a rapid decline of PPARγ1 expression. Because promoter activities were not affected by LPS, we focused on mRNA stability and noticed a decreased mRNA half-life. As RNA stability is often regulated via 3′-untranslated regions (UTRs), we analyzed the impact of the PPARγ-3′-UTR by reporter assays using specific constructs. LPS significantly reduced luciferase activity of the pGL3-PPARγ-3′-UTR, suggesting that PPARγ1 mRNA is destabilized. Deletion or mutation of a potential microRNA-27a/b (miR-27a/b) binding site within the 3′-UTR restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS exposure, partially reversed PPARγ1 mRNA decay, whereas miR-27b overexpression decreased PPARγ1 mRNA content. In addition, LPS further reduced this decay. The functional relevance of miR-27b-dependent PPARγ1 decrease was proven by inhibition or overexpression of miR-27b, which affected LPS-induced expression of the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin (IL)-6. We provide evidence that LPS-induced miR-27b contributes to destabilization of PPARγ1 mRNA. Understanding molecular mechanisms decreasing PPARγ might help to better appreciate inflammatory diseases.

HIF-1 and p53: communication of transcription factors under hypoxia.

Oxygen sensing and reactivity to changes in the concentration of oxygen is a fundamental property of cell physiology. The lack of O2 (hypoxia) is transmitted into many adaptive responses, a process that is largely controlled by a transcription factor known as hypoxia inducible factor‐1 (HIF‐1). More recent reports suggest that besides its traditional regulation via proteasomal degradation other signaling pathways contribute to stability regulation of the HIF‐1α subunit and/or HIF‐1 transactivation. These regulatory circuits allow for the integration of HIF‐1 into scenarios of cell‐survival vs. cell‐death with the rule of the thumb that short‐term mild hypoxia maintains cell viability while prolonged and severe hypoxia provokes cell demise. Cell death pathways are associated with stabilization of the tumor suppressor p53, a response also seen under hypoxic conditions. Here we summarize recent information on accumulation of HIF‐1α and p53 under hypoxia and provide a model to explain the communication between HIF‐1 and p53 under (patho)physiological conditions.

p300 relieves p53-evoked transcriptional repression of hypoxia-inducible factor-1 (HIF-1).

HIF-1 (hypoxia-inducible factor-1), a heterodimeric transcription factor comprising HIF-1alpha and HIF-1beta subunits, serves as a key regulator of metabolic adaptation to hypoxia. HIF-1 activity largely increases during hypoxia by attenuating pVHL (von Hippel-Lindau protein)-dependent ubiquitination and subsequent 26 S-proteasomal degradation of HIF-1alpha. Besides HIF-1, the transcription factor and tumour suppressor p53 accumulates and is activated under conditions of prolonged/severe hypoxia. Recently, the interaction between p53 and HIF-1alpha was reported to evoke HIF-1alpha degradation. Destruction of HIF-1alpha by p53 was corroborated in the present study by using pVHL-deficient RCC4 (renal carcinoma) cells, supporting the notion of a pVHL-independent degradation process. In addition, low p53 expression repressed HIF-1 transactivation without affecting HIF-1alpha protein amount. Establishing that p53-evoked inhibition of HIF-1 reporter activity was relieved upon co-transfection of p300 suggested competition between p53 and HIF-1 for limiting amounts of the shared co-activator p300. This assumption was confirmed by showing competitive binding of in vitro transcription/translation-generated p53 and HIF-1alpha to the CH1 domain of p300 in vitro. We conclude that low p53 expression attenuates HIF-1 transactivation by competing for p300, whereas high p53 expression destroys the HIF-1alpha protein and thereby eliminates HIF-1 reporter activity. Thus once p53 becomes activated under conditions of severe hypoxia/anoxia, it contributes to terminating HIF-1 responses.

Roles of hypoxia‐inducible factor‐1α (HIF‐1α) versus HIF‐2α in the survival of hepatocellular tumor spheroids

Hypoxia‐inducible factors (HIFs) provoke adaptation to hypoxic stress occurring in rapidly growing tumor tissues. Therefore, overexpression of HIF‐1 or HIF‐2 is a common feature in hepatocellular carcinoma but their specific function is still controversially discussed. To analyze HIF function in hypoxia‐induced cell death we created a stable knockdown of HIF‐1α and HIF‐2α in HepG2 cells and generated tumor spheroids as an in vitro hepatocellular carcinoma model. Knockdown of HIF‐1α enhanced expression of HIF‐2α and vice versa. Unexpectedly, knockdown of HIF‐1α or HIF‐2α increased cell viability as well as spheroid size and decreased caspase‐3 activity. Antiapoptotic Bcl‐XL expression increased in both knockdown spheroids, whereas proapoptotic Bax was only reduced in HIF‐1α‐knockdown cells. Furthermore, an HIF‐2α‐knockdown significantly increased Bcl‐2/adenovirus E1B 19 kDa‐interacting protein 3 (BNIP3) expression in an HIF‐1α‐dependent manner. Concomitantly, electron microscopy revealed a substantial increase in autophagosomal structures in HIF‐2α‐knockdown spheroids and mito‐/lysotracker costaining confirmed lysosomal activity of these autophagosomes. Blocking autophagosome maturation using 3‐methyladenine restored cell death in HIF‐2α‐knockdown clones comparable to wildtype cells. Conclusion: An HIF‐1α‐knockdown increases HIF‐2α expression and shifts the balance of Bcl‐2 family members toward survival. The knockdown of HIF‐2α raises autophagic activity and attenuates apoptosis by enhancing HIF‐1α expression. Our data indicate that enhanced expression of one HIF‐isoform causes a survival advantage in hepatocellular carcinoma development. HEPATOLOGY 2010

Glucocorticoid-induced leucine zipper is downregulated in human alveolar macrophages upon Toll-like receptor activation

Induction of the glucocorticoid‐induced leucine zipper (GILZ) by glucocorticoids plays a role in their antiinflammatory action, whereas GILZ expression is reduced under inflammatory conditions. The mechanisms regulating GILZ expression during inflammation, however, have not yet been characterized. Here, we investigated GILZ expression in human alveolar macrophages (AMs) following Toll‐like receptor (TLR) activation. Macrophages were shown to predominantly express GILZ transcript variant 2. Lipopolysaccharide‐treated AMs, THP‐1 cells, and lungs of lipopolysaccharide‐exposed mice displayed decreased GILZ protein and mRNA levels. The effect was strictly dependent on the adapter molecule MyD88, as shown by using specific ligands or a knockdown strategy. Investigations on the functional significance of GILZ downregulation performed by GILZ knockdown revealed a proinflammatory response, as indicated by increased cytokine expression and NF‐κB activity. We found that TLR activation reduced GILZ mRNA stability, which was mediated via the GILZ 3′‐untranslated region. Finally, involvement of the mRNA‐binding protein tristetraprolin (TTP) is suggested, since TTP overexpression or knockdown modulated GILZ expression and TTP was induced in a MyD88‐dependent fashion. Taken together, our data show a MyD88‐ and TTP‐dependent GILZ downreg‐ulation in human macrophages upon TLR activation. Suppression of GILZ is mediated by mRNA destabilization, which might represent a regulatory mechanism in macrophage activation.

Inhibition of GSK3β by indirubins restores HIF-1α accumulation under prolonged periods of hypoxia/anoxia

Hypoxia inducible factor 1 is regulated by the appearance of the HIF‐1α subunit. HIF‐1α is subjected to proteasomal destruction or enhanced protein translation, which requires the phosphatidylinositol 3‐kinase (PI3K)/Akt pathway. We investigated how PI3K/Akt and glycogen synthase kinase 3β (GSK3β) affect HIF‐1α in human RKO cells under prolonged periods of severe hypoxia/anoxia. 16‐ to 32‐h lasting incubations attenuated Akt activity and decreased HIF‐1α protein. This was reproduced by blocking PI3K with LY294002. GSK3β inhibition by indirubins circumvented the effect of hypoxia/anoxia or LY294002 on HIF‐1α. Ruling stability regulation of HIF‐1α protein and/or enhanced transcription of HIF‐1α mRNA via GSK3β inhibition out is suggestive for translational modulation of HIF‐1α under the influence of GSK3β.

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.

Downregulation of programmed cell death 4 by inflammatory conditions contributes to the generation of the tumor promoting microenvironment

Ample evidence has shown key roles of inflammation in tumor promotion and carcinogenesis, and tumor‐associated macrophages are known to promote tumor growth and dissemination. Programmed cell death 4 (Pdcd4) is a novel tumor suppressor, and although various studies have revealed that the functions and expression mechanisms of Pdcd4 in tumor promotion, those in regard to inflammation remain unclear. In the present study, we examined whether inflammatory stimuli regulate Pdcd4 expression. 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) suppressed expression of pdcd4 mRNA in human monocytic cell lines (U937, THP‐1). Similarly, the bacterial endotoxin lipopolysaccharide (LPS) downregulated pdcd4 level in mouse RAW264.7 and peritoneal macrophages. Furthermore, conditioned medium from LPS‐stimulated RAW264.7 macrophages suppressed pdcd4 mRNA in RAW264.7 macrophages, and findings obtained with recombinant tumor necrosis factor‐α (TNF‐α) and TNF‐α‐specific siRNA suggested that TNF‐α partly mediates LPS‐triggered Pdcd4 downregulation via an autocrine mechanism. Specific inhibitors of phosphoinositide‐3‐kinase (PI3K) and c‐jun N‐terminus kinase (JNK) restored LPS‐abolished pdcd4 mRNA. Consistently, in MCF7 mammary carcinoma cells, conditioned medium from TPA‐differentiated/activated U937 cells suppressed pdcd4 mRNA. Additionally, knockdown of pdcd4 in RAW264.7 macrophages using siRNA significantly enhanced LPS‐induced TNF‐α protein production, and interferon‐γ, CC chemokine ligand (Ccl) 1, Ccl20, and interleukin‐10 mRNA expression. These results suggest that Pdcd4 suppresses the induction of these inflammatory mediators. Taken together, loss of Pdcd4 in macrophages may be a critical step in establishing the inflammatory environment while that in tumor cells contributes to tumor progression.

Erioflorin Stabilizes the Tumor Suppressor Pdcd4 by Inhibiting Its Interaction with the E3-ligase β-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 p70S6K1-dependent protein phosphorylation, β-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 β-TrCP1 and Pdcd4 in cell culture and in in vitro binding assays, consequently decreasing ubiquitination and degradation of Pdcd4. Interestingly, while erioflorin stabilized additional β-TrCP-targets (such as IκBα and β-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 β-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 β-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.