Regulation of IL-1β-induced NFκB by hydroxylases links key hypoxic and inflammatory signaling pathways

Hypoxia is a prominent feature of chronically inflamed tissues. Oxygen-sensing hydroxylases control transcriptional adaptation to hypoxia through the regulation of hypoxia-inducible factor (HIF) and nuclear factor ?B (NF-?B), both of which can regulate the inflammatory response. Furthermore, pharmacologic hydroxylase inhibitors reduce inflammation in multiple animal models. However, the underlying mechanism(s) linking hydroxylase activity to inflammatory signaling remains unclear. IL-1ß, a major proinflammatory cytokine that regulates NF-?B, is associated with multiple inflammatory pathologies. We demonstrate that a combination of prolyl hydroxylase 1 and factor inhibiting HIF hydroxylase isoforms regulates IL-1ß-induced NF-?B at the level of (or downstream of) the tumor necrosis factor receptor-associated factor 6 complex. Multiple proteins of the distal IL-1ß-signaling pathway are subject to hydroxylation and form complexes with either prolyl hydroxylase 1 or factor inhibiting HIF. Thus, we hypothesize that hydroxylases regulate IL-1ß signaling and subsequent inflammatory gene expression. Furthermore, hydroxylase inhibition represents a unique approach to the inhibition of IL-1ß-dependent inflammatory signaling

Prolyl hydroxylase-1 regulates hepatocyte apoptosis in an NF-kB-dependent manner

Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1(-/-) hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease.status: publishe

Impairment of angiogenesis by fatty acid synthase inhibition Involves mTOR malonylation

The role of fatty acid synthesis in endothelial cells (ECs) remains incompletely characterized. We report that fatty acid synthase knockdown (FASNKD) in ECs impedes vessel sprouting by reducing proliferation. Endothelial loss of FASN impaired angiogenesis in vivo, while FASN blockade reduced pathological ocular neovascularization, at >10-fold lower doses than used for anti-cancer treatment. Impaired angiogenesis was not due to energy stress, redox imbalance, or palmitate depletion. Rather, FASNKD elevated malonyl-CoA levels, causing malonylation (a post-translational modification) of mTOR at lysine 1218 (K1218). mTOR K-1218 malonylation impaired mTOR complex 1 (mTORC1) kinase activity, thereby reducing phosphorylation of downstream targets (p70S6K/4EBP1). Silencing acetyl-CoA carboxylase 1 (an enzyme producing malonyl-CoA) normalized malonyl-CoA levels and reactivated mTOR in FASNKD ECs. Mutagenesis unveiled the importance of mTOR K1218 malonylation for angiogenesis. This study unveils a novel role of FASN in metabolite signaling that contributes to explaining the anti-angiogenic effect of FASN blockade

NFκB and HIF display synergistic behaviour during hypoxic inflammation

The oxygen-sensitive transcription factor hypoxia inducible factor (HIF) is a key regulator of gene expression during adaptation to hypoxia. Crucially, inflamed tissue often displays regions of prominent hypoxia. Recent studies have shown HIF signalling is intricately linked to that of the pro-inflammatory transcription factor nuclear factor kappa B (NFκB) during hypoxic inflammation. We describe the relative temporal contributions of each to hypoxia-induced inflammatory gene expression and investigate the level of crosstalk between the two pathways using a novel Gaussia princeps luciferase (Gluc) reporter system. Under the control of an active promoter, Gluc is expressed and secreted into the cell culture media, where it can be sampled and measured over time. Thus, Gluc constructs under the control of either HIF or NFκB were used to resolve their temporal transcriptional dynamics in response to hypoxia and to cytokine stimuli, respectively. We also investigated the interactions between HIF and NFκB activities using a construct containing the sequence from the promoter of the inflammatory gene cyclooxygenase 2 (COX-2), which includes functionally active binding sites for both HIF and NFκB. Finally, based on our experimental data, we constructed a mathematical model of the binding affinities of HIF and NFκB to their respective response elements to analyse transcriptional crosstalk. Taken together, these data reveal distinct temporal HIF and NFκB transcriptional activities in response to hypoxic inflammation. Furthermore, we demonstrate synergistic activity between these two transcription factors on the regulation of the COX-2 promoter, implicating a co-ordinated role for both HIF and NFκB in the expression of COX-2 in hypoxic inflammation.Science Foundation Ireland (Grant No.06/CE/B1129)Deposited by bulk impor

Hypercapnia induces cleavage and nuclear localization of relb protein, giving insight into co2sensing and signaling

Carbon dioxide (CO2) is increasingly being appreciated as an intracellular signaling molecule that affects inflammatory and immune responses. Elevated arterial CO2 (hypercapnia) is encountered in a range of clinical conditions, including chronic obstructive pulmonary disease, and as a consequence of therapeutic ventilation in acute respiratory distress syndrome. In patients suffering from this syndrome, therapeutic hypoventilation strategy designed to reduce mechanical damage to the lungs is accompanied by systemic hypercapnia and associated acidosis, which are associated with improved patient outcome. However, the molecular mechanisms underlying the beneficial effects of hypercapnia and the relative contribution of elevated CO2 or associated acidosis to this response remain poorly understood. Recently, a role for the noncanonical NF-kappa B pathway has been postulated to be important in signaling the cellular transcriptional response to CO2. In this study, we demonstrate that in cells exposed to elevated CO2, the NF-kappa B family member RelB was cleaved to a lower molecular weight form and translocated to the nucleus in both mouse embryonic fibroblasts and human pulmonary epithelial cells (A549). Furthermore, elevated nuclear RelB was observed in vivo and correlated with hypercapnia-induced protection against LPS-induced lung injury. Hypercapnia-induced RelB processing was sensitive to proteasomal inhibition by MG-132 but was independent of the activity of glycogen synthase kinase 3 beta or MALT-1, both of which have been previously shown to mediate RelB processing. Taken together, these data demonstrate that RelB is a CO2-sensitive NF-kappa B family member that may contribute to the beneficial effects of hypercapnia in inflammatory diseases of the lung

Hypercapnia induces cleavage and nuclear localization of relb protein, giving insight into co2sensing and signaling

Carbon dioxide (CO2) is increasingly being appreciated as an intracellular signaling molecule that affects inflammatory and immune responses. Elevated arterial CO2 (hypercapnia) is encountered in a range of clinical conditions, including chronic obstructive pulmonary disease, and as a consequence of therapeutic ventilation in acute respiratory distress syndrome. In patients suffering from this syndrome, therapeutic hypoventilation strategy designed to reduce mechanical damage to the lungs is accompanied by systemic hypercapnia and associated acidosis, which are associated with improved patient outcome. However, the molecular mechanisms underlying the beneficial effects of hypercapnia and the relative contribution of elevated CO2 or associated acidosis to this response remain poorly understood. Recently, a role for the noncanonical NF-kappa B pathway has been postulated to be important in signaling the cellular transcriptional response to CO2. In this study, we demonstrate that in cells exposed to elevated CO2, the NF-kappa B family member RelB was cleaved to a lower molecular weight form and translocated to the nucleus in both mouse embryonic fibroblasts and human pulmonary epithelial cells (A549). Furthermore, elevated nuclear RelB was observed in vivo and correlated with hypercapnia-induced protection against LPS-induced lung injury. Hypercapnia-induced RelB processing was sensitive to proteasomal inhibition by MG-132 but was independent of the activity of glycogen synthase kinase 3 beta or MALT-1, both of which have been previously shown to mediate RelB processing. Taken together, these data demonstrate that RelB is a CO2-sensitive NF-kappa B family member that may contribute to the beneficial effects of hypercapnia in inflammatory diseases of the lung

Small Ubiquitin-related Modifier (SUMO)-1 Promotes Glycolysis in Hypoxia*

Under conditions of hypoxia, most eukaryotic cells undergo a shift in metabolic strategy, which involves increased flux through the glycolytic pathway. Although this is critical for bioenergetic homeostasis, the underlying mechanisms have remained incompletely understood. Here, we report that the induction of hypoxia-induced glycolysis is retained in cells when gene transcription or protein synthesis are inhibited suggesting the involvement of additional post-translational mechanisms. Post-translational protein modification by the small ubiquitin related modifier-1 (SUMO-1) is induced in hypoxia and mass spectrometric analysis using yeast cells expressing tap-tagged Smt3 (the yeast homolog of mammalian SUMO) revealed hypoxia-dependent modification of a number of key glycolytic enzymes. Overexpression of SUMO-1 in mammalian cancer cells resulted in increased hypoxia-induced glycolysis and resistance to hypoxia-dependent ATP depletion. Supporting this, non-transformed cells also demonstrated increased glucose uptake upon SUMO-1 overexpression. Conversely, cells overexpressing the de-SUMOylating enzyme SENP-2 failed to demonstrate hypoxia-induced glycolysis. SUMO-1 overexpressing cells demonstrated focal clustering of glycolytic enzymes in response to hypoxia leading us to hypothesize a role for SUMOylation in promoting spatial re-organization of the glycolytic pathway. In summary, we hypothesize that SUMO modification of key metabolic enzymes plays an important role in shifting cellular metabolic strategies toward increased flux through the glycolytic pathway during periods of hypoxic stress

The Cancer Cell Oxygen Sensor PHD2 Promotes Metastasis via Activation of Cancer-Associated Fibroblasts.

Several questions about the role of the oxygen sensor prolyl-hydroxylase 2 (PHD2) in cancer have not been addressed. First, the role of PHD2 in metastasis has not been studied in a spontaneous tumor model. Here, we show that global PHD2 haplodeficiency reduced metastasis without affecting tumor growth. Second, it is unknown whether PHD2 regulates cancer by affecting cancer-associated fibroblasts (CAFs). We show that PHD2 haplodeficiency reduced metastasis via two mechanisms: (1) by decreasing CAF activation, matrix production, and contraction by CAFs, an effect that surprisingly relied on PHD2 deletion in cancer cells, but not in CAFs; and (2) by improving tumor vessel normalization. Third, the effect of concomitant PHD2 inhibition in malignant and stromal cells (mimicking PHD2 inhibitor treatment) is unknown. We show that global PHD2 haplodeficiency, induced not only before but also after tumor onset, impaired metastasis. These findings warrant investigation of PHD2's therapeutic potential