RESUMEN
In solid tumors, tumor-associated macrophages (TAMs) commonly accumulate within hypoxic areas. Adaptations to such environments evoke transcriptional changes by the hypoxia-inducible factors (HIFs). While HIF-1α is ubiquitously expressed, HIF-2α appears tissue-specific with consequences of HIF-2α expression in TAMs only being poorly characterized. An E0771 allograft breast tumor model revealed faster tumor growth in myeloid HIF-2α knockout (HIF-2αLysM-/- ) compared with wildtype (wt) mice. In an RNA-sequencing approach of FACS sorted wt and HIF-2α LysM-/- TAMs, serine protease inhibitor, Kunitz type-1 ( Spint1) emerged as a promising candidate for HIF-2α-dependent regulation. We validated reduced Spint1 messenger RNA expression and concomitant Spint1 protein secretion under hypoxia in HIF-2α-deficient bone marrow-derived macrophages (BMDMs) compared with wt BMDMs. In line with the physiological function of Spint1 as an inhibitor of hepatocyte growth factor (HGF) activation, supernatants of hypoxic HIF-2α knockout BMDMs, not containing Spint1, were able to release proliferative properties of inactive pro-HGF on breast tumor cells. In contrast, hypoxic wt BMDM supernatants containing abundant Spint1 amounts failed to do so. We propose that Spint1 contributes to the tumor-suppressive function of HIF-2α in TAMs in breast tumor development.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Neoplasias/genética , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Microambiente Tumoral/genética , Aloinjertos , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Factor de Crecimiento de Hepatocito/genética , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Glicoproteínas de Membrana/genética , Ratones , Neoplasias/patología , ARN MensajeroRESUMEN
Cell stress such as hypoxia elicits adaptive responses, also on the level of mitochondria, and in part is mediated by the hypoxia-inducible factor (HIF) 1α. Adaptation of mitochondria towards acute hypoxic conditions is reasonably well understood, while regulatory mechanisms, especially of respiratory chain assembly factors, under chronic hypoxia remains elusive. One of these assembly factors is transmembrane protein 126B (TMEM126B). This protein is part of the mitochondrial complex I assembly machinery. We identified changes in complex I abundance under chronic hypoxia, in association with impaired substrate-specific mitochondrial respiration. Complexome profiling of isolated mitochondria of the human leukemia monocytic cell line THP-1 revealed HIF-1α-dependent deficits in complex I assembly and mitochondrial complex I assembly complex (MCIA) abundance. Of all mitochondrial MCIA members, we proved a selective HIF-1-dependent decrease of TMEM126B under chronic hypoxia. Mechanistically, HIF-1α induces the E3-ubiquitin ligase F-box/WD repeat-containing protein 1A (ß-TrCP1), which in turn facilitates the proteolytic degradation of TMEM126B. Attenuating a functional complex I assembly appears critical for cellular adaptation towards chronic hypoxia and is linked to destruction of the mitochondrial assembly factor TMEM126B.
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Mitocondrias/metabolismo , Consumo de Oxígeno , Secuencia de Aminoácidos , Hipoxia de la Célula , Línea Celular Tumoral , Complejo I de Transporte de Electrón/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/genética , Proteolisis , Interferencia de ARN , Células THP-1RESUMEN
Macrophages are known for their versatile role in biology. They sense and clear structures that contain exogenous or endogenous pathogen-associated molecular patterns. This process is tightly linked to the production of a mixture of potentially harmful oxidants and cytokines. Their inherent destructive behavior is directed against foreign material or structures of 'altered self', which explains the role of macrophages during innate immune reactions and inflammation. However, there is also another side of macrophages when they turn into a tissue regenerative, pro-resolving, and healing phenotype. Phenotype changes of macrophages are termed macrophage polarization, representing a continuum between classical and alternative activation. Macrophages as the dominating producers of superoxide/hydrogen peroxide and nitric oxide are not only prone to oxidative modifications but also to more subtle signaling properties of redox-active molecules conveying redox regulation. We review basic concepts of the enzymatic nitric oxide and superoxide production within macrophages, refer to their unique chemical reactions and outline biological consequences not only for macrophage biology but also for their communication with cells in the microenvironment. These considerations link hypoxia to the NO system, addressing feedforward as well as feedback circuits. Moreover, we summarize the role of redox-signaling affecting epigenetics and reflect the central role of mitochondrial-derived oxygen species in inflammation. To better understand the diverse functions of macrophages during initiation as well as resolution of inflammation and to decode their versatile roles during innate and adaptive immunity with the entire spectrum of cell protective towards cell destructive activities we need to appreciate the signaling properties of redox-active species. Herein we discuss macrophage responses in terms of nitric oxide and superoxide formation with the modulating impact of hypoxia.
Asunto(s)
Macrófagos/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Transducción de Señal , Animales , Cromatina/genética , Cromatina/metabolismo , Humanos , Hipoxia/metabolismo , Inmunidad , Inflamación/etiología , Inflamación/metabolismo , Macrófagos/inmunología , Mitocondrias/metabolismo , Células Mieloides/inmunología , Células Mieloides/metabolismo , NADPH Oxidasas/metabolismo , Óxido Nítrico/metabolismoRESUMEN
The RNA-binding protein HuR promotes tumor growth by affecting proliferation, metastasis, apoptosis, and angiogenesis. Although immune cells, especially tumor-associated macrophages, are critical components of the tumor stroma, the influence of HuR in tumors on the recruitment of immune cells remains poorly understood. In the present study, we, therefore, aimed to elucidate the impact of tumor cell HuR on the interaction between tumor cells and macrophages. To this end, we stably depleted HuR in human MCF-7 breast cancer cells. We found that HuR-deficient cells not only showed reduced proliferation, they further expressed elevated levels of the chemokine CCL5. HuR-dependent repression of CCL5 was neither caused by altered CCL5 mRNA stability, nor by changes in CCL5 translation. Instead, loss of HuR augmented transcription of CCL5, which was mediated via an interferon-stimulated response element in the CCL5 promoter. Furthermore, HuR depletion enhanced macrophage recruitment into MCF-7 tumor spheroids, an effect which was completely lost upon neutralization of CCL5. HuR expression further negatively correlated with CCL5 expression and macrophage appearance in a cohort of breast tumors. Thus, while HuR is well-characterized to support various pro-tumorigenic features in tumor cells, we provide evidence that it limits the recruitment of macrophages into tumors by repressing CCL5. As macrophage infiltration is associated with poor prognosis, our findings underline the highly cell-type and context specific role of HuR in tumorigenesis.
Asunto(s)
Neoplasias de la Mama/genética , Quimiocina CCL5/genética , Proteína 1 Similar a ELAV/genética , Regulación Neoplásica de la Expresión Génica , Macrófagos/metabolismo , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Células Cultivadas , Quimiocina CCL5/metabolismo , Técnicas de Cocultivo , Estudios de Cohortes , Proteína 1 Similar a ELAV/metabolismo , Femenino , Humanos , Células MCF-7 , Macrófagos/citología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esferoides Celulares/metabolismoRESUMEN
Tumors are composed of tumor cells, nonmalignant cells, and the vascular system. Among them is intense communication via cell-cell contact-dependent mechanisms and/or soluble messengers. In the tumor microenvironment cells often face a certain degree of oxygen and nutrient deprivation. Hypoxic stress alters the metabolism of tumor cells but also of macrophages, as one dominating immune cell population in most solid tumors, with subsequent changes in the microenvironment. This alters the phenotype and metabolism of macrophages, to induce a tumor-promoting reprogramming. Nutrient stress also provokes autophagy to guarantee cell survival or, if overwhelmed, to exit toward cell demise. Death of tumor cells turned out as a communicative system attracting macrophages and directing their phenotype. Depending on the mode of tumor cell death macrophage polarization ranges from the extremes of pro-inflammatory activation toward anti-inflammatory/immuno-suppressive activation. Here we discuss how hypoxia and cell death adds the cross-talk between cancer cells and macrophages.
Asunto(s)
Hipoxia/metabolismo , Macrófagos/metabolismo , Neoplasias/metabolismo , Microambiente Tumoral , Animales , Muerte Celular , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismoRESUMEN
Renal mesangial cells are regarded as main players in glomerular inflammatory diseases. To investigate a possible crosstalk between inflammatory and hypoxia-driven signaling processes, we stimulated cultured mouse mesangial cells with different inflammatory agents and analyzed the expression of prolyl hydroxylase domain containing proteins (PHDs), the main regulators of hypoxia-inducible factor (HIF) stability. Administration of IL-1ß (1 nM) and TNF-α (1 nM), a combination further referred to as cytokine mix (CM), resulted in a fivefold increase in PHD3 but not PHD1 and PHD2 mRNA expression compared to untreated controls. In contrast, a combination of IL-1ß, TNF-α with lipopolysaccharide (10 µg/ml), and interferon-γ (20 ng/ml) designated as CM+ showed a high (60-fold) induction of PHD3 and a moderate (twofold) induction of PHD2 mRNA expression. Interestingly, CM+ but not CM induced the expression of inducible NO synthase and endogenously produced NO was responsible for the immense induction of PHD3 in mesangial cells treated with CM+. We found that CM+ affected PHD3 expression mainly via the NO/HIF axis, whereas PHD3 regulation by CM occurred in a NF-κB-dependent manner. In turn, silencing of PHD3 expression resulted in a decrease in the mRNA expression of ICAM-1, MIP-2, MCP-1, and CXCL-10, which are under control of NF-κB. In a rat model of mesangio-proliferative glomerulonephritis, PHD3 mRNA and protein expression was markedly induced and this effect was nearly abolished when rats were treated with the iNOS-specific inhibitor L-NIL, thus confirming our findings also in vivo. KEY MESSAGE: PHD3 expression induced by cytokines is NF-κB dependent in mesangial cells. Endogenously produced NO further augments PHD3 expression via HIF-1α. PHD3 expression is induced by NO in anti-Thy-1 glomerulonephritis.
Asunto(s)
Glomerulonefritis/genética , Óxido Nítrico/inmunología , Procolágeno-Prolina Dioxigenasa/genética , Regulación hacia Arriba , Animales , Células Cultivadas , Glomerulonefritis/inmunología , Glomerulonefritis/patología , Humanos , Interleucina-1beta/inmunología , Células Mesangiales/inmunología , Células Mesangiales/metabolismo , Células Mesangiales/patología , Ratones , Ratones Endogámicos C57BL , FN-kappa B/inmunología , Procolágeno-Prolina Dioxigenasa/inmunología , ARN Mensajero/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
SIGNIFICANCE: Leukocytes and especially macrophages are a major cellular constituent of the tumor mass. The tumor microenvironment not only determines their activity but in turn these cells also contribute to tumor initiation and progression. Recent Advances: Proinflammatory stimulated macrophages upregulate inducible nitric oxide synthase (NOS2) and produce high steady-state NO concentrations. NO provokes tumor cell death by initiating apoptosis and/or necrosis. Mechanisms may comprise p53 accumulation, immunestimulatory activities, and an increased efficacy of chemo- and/or radiotherapy. However, the potential cytotoxic activity of macrophages often is compromised in the tumor microenvironment and instead a protumor activity of macrophages dominates. Contributing factors are signals generated by viable and dying tumor cells, attraction and activation of myeloid-derived suppressor cells, and hypoxia. Limited oxygen availability not only attenuates NOS2 activity but also causes accumulation of hypoxia-inducible factors 1 and 2 (HIF-1/HIF-2). Activation of the HIF system is tightly linked to NO formation and affects the expression of macrophage phenotype markers that in turn add to tumor progression. CRITICAL ISSUES: To make use of the cytotoxic arsenal of activated macrophages directed against tumor cells, it will be critical to understand how, when, and where these innate immune responses are blocked and whether it will be possible to reinstall their full capacity to kill tumor cells. FUTURE DIRECTIONS: Low-dose irradiation or proinflammatory activation of macrophages in the tumor microenvironment may open options to boost NOS2 expression and activity and to initiate immunestimulatory features of NO that may help to restrict tumor growth. Antioxid. Redox Signal. 26, 1023-1043.
Asunto(s)
Leucocitos/metabolismo , Macrófagos/metabolismo , Neoplasias/inmunología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Animales , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Activación de Macrófagos , Macrófagos/patología , Neoplasias/metabolismo , Óxido Nítrico/metabolismo , Microambiente TumoralRESUMEN
Hypoxia, by activating transcription factors induces transcription of some genes but it also reduces mRNA synthesis by mechanisms that are poorly defined. Activation of human macrophages with interleukin (IL)-4 showed that up-regulation of some IL-4 target genes was reduced when macrophages were incubated at 1% oxygen. Hypoxia impaired induction of chemokine (C-C motif) ligand 18 (CCL18), although IL-4-induced DNA binding of the transcription factor STAT6 remained intact. In contrast, induction of serine peptidase inhibitor, Kunitz type (SPINT)2, another IL-4/STAT6 target gene, was not affected by hypoxia. The repressive histone mark histone 3 lysine 27 trimethylation (H3K27me3), known to prevent chromatin remodelling and transcription, was removed from the SPINT2 but not the CCL18 gene locus under hypoxia or dimethyloxalylglycine-treatment. The H3K27me3 demethylase JMJD3 was required for CCL18 gene induction but dispensable for induction of SPINT2. Our data indicate that hypoxic inhibition of JMJD3 activity reduces demethylation of H3K27me3, nucleosome removal, and hence induction of the STAT6 target gene CCL18, while induction of other STAT6-inducible genes such as SPINT2 remained unaffected by JMJD3. In contrast to mouse MΦ in human cells JMJD3 is not recruited by transcription factors like IRF4, KL4, or PPARγ to convey specificity in gene induction.
Asunto(s)
Diferenciación Celular/genética , Quimiocinas CC/genética , Histona Demetilasas con Dominio de Jumonji/genética , Glicoproteínas de Membrana/genética , Factor de Transcripción STAT6/genética , Animales , Hipoxia de la Célula/genética , Metilación de ADN/genética , Regulación del Desarrollo de la Expresión Génica , Histona Demetilasas/genética , Humanos , Interleucina-4/genética , Ratones , Nucleosomas/genética , Activación Transcripcional/genéticaRESUMEN
Hypoxia-inducible factor-1α (HIF-1α), which accumulates in mammalian host organisms during infection, supports the defense against microbial pathogens. However, whether and to what extent HIF-1α expressed by myeloid cells contributes to the innate immune response against Leishmania major parasites is unknown. We observed that Leishmania-infected humans and L. major-infected C57BL/6 mice exhibited substantial amounts of HIF-1α in acute cutaneous lesions. In vitro, HIF-1α was required for leishmanicidal activity and high-level NO production by IFN-γ/LPS-activated macrophages. Mice deficient for HIF-1α in their myeloid cell compartment had a more severe clinical course of infection and increased parasite burden in the skin lesions compared with wild-type controls. These findings were paralleled by reduced expression of type 2 NO synthase by lesional CD11b+ cells. Together, these data illustrate that HIF-1α is required for optimal innate leishmanicidal immune responses and, thereby, contributes to the cure of cutaneous leishmaniasis.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Células Mieloides/metabolismo , Piel/parasitología , Animales , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inmunidad Innata , Interferón gamma/farmacología , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/patología , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Macrófagos/parasitología , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/genética , Carga de Parásitos , Piel/patologíaRESUMEN
Post-translational modification of proteins with ubiquitin-like SUMO modifiers is a tightly regulated and highly dynamic process. The SENP family of SUMO-specific isopeptidases comprises six cysteine proteases. They are instrumental in counterbalancing SUMO conjugation, but their regulation is not well understood. We demonstrate that in hypoxic cell extracts, the catalytic activity of SENP family members, in particular SENP1 and SENP3, is inhibited in a rapid and fully reversible process. Comparative mass spectrometry from normoxic and hypoxic cells defines a subset of hypoxia-induced SUMO1 targets, including SUMO ligases RanBP2 and PIAS2, glucose transporter 1, and transcriptional regulators. Among the most strongly induced targets, we identified the transcriptional co-repressor BHLHE40, which controls hypoxic gene expression programs. We provide evidence that SUMOylation of BHLHE40 is reversed by SENP1 and contributes to transcriptional repression of the metabolic master regulator gene PGC-1α. We propose a pathway that connects oxygen-controlled SENP activity to hypoxic reprogramming of metabolism.
Asunto(s)
Liasas de Carbono-Nitrógeno/metabolismo , Transducción de Señal , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Biocatálisis , Hipoxia de la Célula , Proteínas Co-Represoras/metabolismo , Cisteína Endopeptidasas/metabolismo , Activación Enzimática , Células HeLa , Humanos , Especificidad por Sustrato , SumoilaciónRESUMEN
Activation of hypoxia-inducible factor (HIF) and macrophage infiltration of solid tumors independently promote tumor progression. As little is known how myeloid HIF affects tumor development, we injected the polycyclic aromatic hydrocarbon (PAH) and procarcinogen 3-methylcholanthrene (MCA; 100 µg/100 µl) subcutaneously into myeloid-specific Hif-1α and Hif-2α knockout mice (C57BL/6J) to induce fibrosarcomas (n = 16). Deletion of Hif-1α but not Hif-2α in macrophages diminished tumor outgrowth in the MCA-model. While analysis of the tumor initiation phase showed comparable inflammation after MCA-injection, metabolism of MCA was impaired in the absence of Hif-1α. An ex vivo macrophage/fibroblast coculture recapitulated reduced DNA damage after MCA-stimulation in fibroblasts of cocultures with Hif-1α LysM-/- macrophages compared to wild type macrophages. A loss of myeloid Hif-1α decreased RNA levels of arylhydrocarbon receptor (AhR)/arylhydrocarbon receptor nuclear translocator (ARNT) targets such as Cyp1a1 because of reduced Arnt but unchanged Ahr expression. Cocultures using Hif-1α LysM-/- macrophages stimulated with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA; 2 µg/ml) also attenuated a DNA damage response in fibroblasts, while the DNA damage-inducing metabolite DMBA-trans-3,4-dihydrodiol remained effective in the absence of Hif-1α. In chemical-induced carcinogenesis, HIF-1α in macrophages maintains ARNT expression to facilitate PAH-biotransformation. This implies a metabolic activation of PAHs in stromal cells, i.e. myeloid-derived cells, to be crucial for tumor initiation.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Macrófagos/metabolismo , Neoplasias Experimentales/patología , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Carcinógenos/toxicidad , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/patología , Metilcolantreno/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/metabolismo , Hidrocarburos Policíclicos Aromáticos/toxicidad , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
Pro-inflammatory cytokines secreted by adipose tissue macrophages (ATMs) contribute to chronic low-grade inflammation and obesity-induced insulin resistance. Recent studies have shown that adipose tissue hypoxia promotes an inflammatory phenotype in ATMs. However, our understanding of how hypoxia modulates the response of ATMs to free fatty acids within obese adipose tissue is limited. We examined the effects of hypoxia (1% O2) on the pro-inflammatory responses of human monocyte-derived macrophages to the saturated fatty acid palmitate. Compared with normoxia, hypoxia significantly increased palmitate-induced mRNA expression and protein secretion of IL-6 and IL-1ß. Although palmitate-induced endoplasmic reticulum stress and nuclear factor κB pathway activation were not enhanced by hypoxia, hypoxia increased the activation of JNK and p38 mitogen-activated protein kinase signaling in palmitate-treated cells. Inhibition of JNK blocked the hypoxic induction of pro-inflammatory cytokine expression, whereas knockdown of hypoxia-induced transcription factors HIF-1α and HIF-2α alone or in combination failed to reduce IL-6 and only modestly reduced IL-1ß gene expression in palmitate-treated hypoxic macrophages. Enhanced pro-inflammatory cytokine production and JNK activity under hypoxia were prevented by inhibiting reactive oxygen species generation. In addition, silencing of dual-specificity phosphatase 16 increased normoxic levels of IL-6 and IL-1ß and reduced the hypoxic potentiation in palmitate-treated macrophages. The secretome of hypoxic palmitate-treated macrophages promoted IL-6 and macrophage chemoattractant protein 1 expression in primary human adipocytes, which was sensitive to macrophage JNK inhibition. Our results reveal that the coexistence of hypoxia along with free fatty acids exacerbates macrophage-mediated inflammation.
Asunto(s)
Inflamación/patología , Macrófagos/patología , Palmitatos/farmacología , Acetilcisteína/farmacología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Citocinas/biosíntesis , Citocinas/genética , Fosfatasas de Especificidad Dual/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Compuestos Organofosforados/farmacología , Oxígeno/metabolismo , Fosforilación/efectos de los fármacos , Piperidinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Macrophages respond to the Th2 cytokine IL-4 with elevated expression of arachidonate 15-lipoxygenase (ALOX15). Although IL-4 signaling elicits anti-inflammatory responses, 15-lipoxygenase may either support or inhibit inflammatory processes in a context-dependent manner. AMP-activated protein kinase (AMPK) is a metabolic sensor/regulator that supports an anti-inflammatory macrophage phenotype. How AMPK activation is linked to IL-4-elicited gene signatures remains unexplored. Using primary human macrophages stimulated with IL-4, we observed elevated ALOX15 mRNA and protein expression, which was attenuated by AMPK activation. AMPK activators, e.g. phenformin and aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside inhibited IL-4-evoked activation of STAT3 while leaving activation of STAT6 and induction of typical IL-4-responsive genes intact. In addition, phenformin prevented IL-4-induced association of STAT6 and Lys-9 acetylation of histone H3 at the ALOX15 promoter. Activating AMPK abolished cellular production of 15-lipoxygenase arachidonic acid metabolites in IL-4-stimulated macrophages, which was mimicked by ALOX15 knockdown. Finally, pretreatment of macrophages with IL-4 for 48 h increased the mRNA expression of the proinflammatory cytokines IL-6, IL-12, CXCL9, and CXCL10 induced by subsequent stimulation with lipopolysaccharide. This response was attenuated by inhibition of ALOX15 or activation of AMPK during incubation with IL-4. In conclusion, limiting ALOX15 expression by AMPK may promote an anti-inflammatory phenotype of IL-4-stimulated human macrophages.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Regulación Enzimológica de la Expresión Génica , Interleucina-4/metabolismo , Macrófagos/enzimología , Antiinflamatorios/química , Quimiocina CXCL10/metabolismo , Quimiocina CXCL9/metabolismo , Perfilación de la Expresión Génica , Humanos , Inflamación/metabolismo , Subunidad p35 de la Interleucina-12/metabolismo , Interleucina-6/metabolismo , Monocitos/citología , Fagocitos/metabolismo , Fenotipo , ARN Interferente Pequeño/metabolismo , Factor de Transcripción STAT3/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Under inflammatory conditions or during tumor progression macrophages acquire distinct phenotypes, with factors of the microenvironment such as hypoxia and transforming growth factor ß (TGFß) shaping their functional plasticity. TGFß is among the factors causing alternative macrophage activation, which contributes to tissue regeneration and thus, resolution of inflammation but may also provoke tumor progression. However, the signal crosstalk between TGFß and hypoxia is ill defined. RESULTS: Exposing human primary macrophages to TGFß elicited a rapid SMAD2/SMAD3 phosphorylation. This early TGFß-signaling remained unaffected by hypoxia. However, with prolonged exposure periods to TGFß/hypoxia the expression of SMAD2 declined because of decreased protein stability. In parallel, hypoxia increased mRNA and protein amount of the calpain regulatory subunit, with the further notion that TGFß/hypoxia elicited calpain activation. The dual specific proteasome/calpain inhibitor MG132 and the specific calpain inhibitor 1 rescued SMAD2 degradation, substantiating the ability of calpain to degrade SMAD2. Decreased SMAD2 expression reduced TGFß transcriptional activity of its target genes thrombospondin 1, dystonin, and matrix metalloproteinase 2. CONCLUSIONS: Hypoxia interferes with TGFß signaling in macrophages by calpain-mediated proteolysis of the central signaling component SMAD2.
RESUMEN
BACKGROUND INFORMATION: Tumour-associated lymphangiogenesis was identified as an important clinical determinant for the prognosis of hepatocellular carcinoma (HCC) and significantly influences patient survival. However, in this context, little is known about regulation of lymphangiogenesis by hypoxia-inducible factors (HIF). In HCC, mainly HIF-1α was positively correlated with lymphatic invasion and metastasis, whereas a defined role of HIF-2α is missing. RESULTS: We created a stable knockdown (k/d) of HIF-1α and HIF-2α in HepG2 cells and generated co-cultures of HepG2 spheroids with embryonic bodies. This constitutes an in vitro tumour model mimicking the cancer microenvironment and allows addressing the role of distinct HIF isoforms in regulating HCC lymphangiogenesis. In co-cultures with a HIF-2α k/d, lymphangiogenesis was significantly increased, whereas the k/d of HIF-1α showed no effect. The HIF-2α-dependent lymphangiogenic phenotype was confirmed in vivo using matrigel plug assays with supernatants of HIF-2α k/d HepG2 cells. We identified and verified insulin-like growth factor binding protein 1 (IGFBP1) as a HIF-2α target gene. The potential of HepG2 cells to induce lymphangiogenesis in two independent functional assays was significantly enhanced either by a k/d of HIF-2α or by silencing IGFBP1. Moreover, we confirmed IGF as a potent pro-lymphatic growth factor with IGFBP1 being its negative modulator. CONCLUSIONS: We propose that HIF-2α acts as an important negative regulator of hepatic lymphangiogenesis in vitro and in vivo by inducing IGFBP1 and thus, interfering with IGF signalling. Therefore, HIF-2α may constitute a critical target in HCC therapy.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Carcinoma Hepatocelular/genética , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Neoplasias Hepáticas/genética , Linfangiogénesis/genética , Regulación hacia Arriba/genética , Animales , Línea Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Femenino , Células Hep G2 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Metástasis Linfática/genética , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Tumor associated macrophages (TAMs) are known to support tumor progression and their accumulation is generally associated with poor prognosis. The shift from a tumor-attacking to a tumor-supportive macrophage phenotype is based on an educational program that, at least in part, is initiated by apoptotic tumor cells. AIMS: We explored the macrophage phenotype shift during tumor progression by analyzing the macrophage NO-output system and examining potential NO targets. METHODS: Biochemical and Molecular Biology-orientated cell culture experiments, in part using 3d-tumor spheroid models as well as animal experiments were used. RESULTS: Apoptotic cells polarize macrophages towards a healing, tumor-supportive phenotype. Soluble mediators released from apoptotic cells, among them the lipid sphingosine-1-phosphate (S1P), cause expression of arginase 2 in macrophages, thereby lowering citrulline/NO formation but enhancing ornithine production. Mechanistically, this is achieved via the S1P2 receptor and the CRE (cAMP-response element) binding site in the arginase 2 promoter. Reduced NO-formation is also seen in ex vivo macrophages from a xenograft model allowing restricted vs. unrestricted tumor growth based on tumor-associated S1P-formation. The theoretical ability of NO to target hypoxia-inducible factor-1 (HIF-1) and jumonji histone demethylases (JHDMs) in cells of the tumor microenvironment will be discussed in light of the iNOS/arginase balance. Moreover, data on the importance of HIF-1 in macrophages for their interaction with tumor cells, polarization, and angiogenic potential will be presented. CONCLUSIONS: We hypothesize that apoptotic death of tumor cells and associated macrophage activation facilitates the progression of malignant disease. The macrophage polarization program affects the NO-output system and the capacity of macrophages to support or restrict tumor growth.
Asunto(s)
Macrófagos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Óxido Nítrico/metabolismo , Microambiente Tumoral , Animales , Hipoxia de la Célula , Humanos , Macrófagos/patología , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Óxido Nítrico/genéticaRESUMEN
Macrophages (MΦ) often accumulate in hypoxic areas, where they significantly influence disease progression. Anti-inflammatory cytokines, such as IL-10, generate alternatively activated macrophages that support tumor growth. To understand how alternative activation affects the transcriptional profile of hypoxic macrophages, we globally mapped binding sites of hypoxia-inducible factor (HIF)-1α and HIF-2α in primary human monocyte-derived macrophages prestimulated with IL-10. 713 HIF-1 and 795 HIF-2 binding sites were identified under hypoxia. Pretreatment with IL-10 altered the binding pattern, with 120 new HIF-1 and 188 new HIF-2 binding sites emerging. HIF-1 binding was most prominent in promoters, while HIF-2 binding was more abundant in enhancer regions. Comparison of ChIP-seq data obtained in other cells revealed a highly cell type specific binding of HIF. In MΦ HIF binding occurred preferentially in already active enhancers or promoters. To assess the roles of HIF on gene expression, primary human macrophages were treated with siRNA against HIF-1α or HIF-2α, followed by genome-wide gene expression analysis. Comparing mRNA expression to the HIF binding profile revealed a significant enrichment of hypoxia-inducible genes previously identified by ChIP-seq. Analysis of gene expression under hypoxia alone and hypoxia/IL-10 showed the enhanced induction of a set of genes including PLOD2 and SLC2A3, while another group including KDM3A and ADM remained unaffected or was reduced by IL-10. Taken together IL-10 influences the DNA binding pattern of HIF and the level of gene induction.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Hipoxia de la Célula/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Interleucina-10/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión , Regulación de la Expresión Génica , Genoma Humano , Transportador de Glucosa de Tipo 3/biosíntesis , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Interleucina-10/administración & dosificación , Macrófagos/metabolismo , Macrófagos/patología , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/biosíntesisRESUMEN
Macrophages play important roles in many diseases and are frequently found in hypoxic areas. A chronic hypoxic microenvironment alters global cellular protein expression, but molecular details remain poorly understood. Although hypoxia-inducible factor (HIF) is an established transcription factor allowing adaption to acute hypoxia, responses to chronic hypoxia are more complex. Based on a two-dimensional differential gel electrophoresis (2D-DIGE) approach, we aimed to identify proteins that are exclusively expressed under chronic but not acute hypoxia (1% O2). One of the identified proteins was cathepsin B (CTSB), and a knockdown of either HIF-1α or -2α in primary human macrophages pointed to an HIF-2α dependency. Although chromatin immunoprecipitation (ChIP) experiments confirmed HIF-2 binding to a CTSB enhancer in acute hypoxia, an increase of CTSB mRNA was evident only under chronic hypoxia. Along those lines, CTSB mRNA stability increased at 48 h but not at 8 h of hypoxia. However, RNA stability at 8 h of hypoxia was enhanced by a knockdown of tristetraprolin (TTP). Inactivation of TTP under prolonged hypoxia was facilitated by c-Jun N-terminal kinase (JNK), and inhibition of this kinase lowered CTSB mRNA levels and stability. We postulate a TTP-dependent mechanism to explain delayed expression of CTSB under chronic hypoxia.
Asunto(s)
Catepsina B/metabolismo , Hipoxia de la Célula/genética , Macrófagos/metabolismo , Estabilidad del ARN/genética , Tristetraprolina/metabolismo , Catepsina B/inmunología , Línea Celular , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/inmunología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Macrófagos/inmunología , ARN Mensajero/genética , ARN Mensajero/inmunología , Tristetraprolina/inmunologíaRESUMEN
Hypoxia promotes progression of hepatocellular carcinoma (HCC), not only affecting tumor cell proliferation and invasion, but also angiogenesis and thus, increasing the risk of metastasis. Hypoxia inducible factors (HIF)-1α and -2α cause adaptation of tumors to hypoxia, still with uncertainties towards the angiogenic switch. We created a stable knockdown of HIF-1α and HIF-2α in HepG2 cells and generated cocultures of HepG2 spheroids with embryonic bodies as an in vitro tumor model mimicking the cancer microenvironment. The naturally occuring oxygen and nutrient gradients within the cocultures allow us to question the role of distinct HIF isoforms in regulating HCC angiogenesis. In cocultures with a HIF-2α knockdown, angiogenesis was attenuated, while the knockdown of HIF-1α was without effect. Microarray analysis identified plasminogen activator inhibitor 1 (PAI-1) as a HIF-2α target gene in HepG2 cells. The knockdown of PAI-1 in HepG2 cells also lowered angiogenesis. Blocking plasmin, the downstream target of PAI-1, with aprotinin in HIF-2α knockdown (k/d) cells proved a cause-effect relation and restored angiogenesis, with no effect on control cocultures. Suggestively, HIF-2α increases PAI-1 to lower concentrations of active plasmin, thereby supporting angiogenesis. We conclude that the HIF-2α target gene PAI-1 favors the angiogenic switch in HCC.