Macrophage HIF-2α regulates tumor-suppressive Spint1 in the tumor microenvironment.

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.

Degradation of the mitochondrial complex I assembly factor TMEM126B under chronic hypoxia.

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.

Myeloid Cell-Derived HIF-1α Promotes Control of Leishmania major.

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.

Hypoxia Potentiates Palmitate-induced Pro-inflammatory Activation of Primary Human Macrophages.

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.

Hypoxic inhibition of JMJD3 reduces H3K27me3 demethylation and induction of the STAT6 target gene CCL18.

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.

The RNA-binding protein HuR inhibits expression of CCL5 and limits recruitment of macrophages into tumors.

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.

AMP-activated protein kinase suppresses arachidonate 15-lipoxygenase expression in interleukin 4-polarized human macrophages.

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.

Genome-wide identification of hypoxia-inducible factor-1 and -2 binding sites in hypoxic human macrophages alternatively activated by IL-10.

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.

HIF-2α attenuates lymphangiogenesis by up-regulating IGFBP1 in hepatocellular carcinoma.

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.

HIF-2alpha-dependent PAI-1 induction contributes to angiogenesis in hepatocellular carcinoma.

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.

Vitamin D promotes vascular regeneration.

BACKGROUND: Vitamin D deficiency in humans is frequent and has been associated with inflammation. The role of the active hormone 1,25-dihydroxycholecalciferol (1,25-dihydroxy-vitamin D3; 1,25-VitD3) in the cardiovascular system is controversial. High doses induce vascular calcification; vitamin D3 deficiency, however, has been linked to cardiovascular disease because the hormone has anti-inflammatory properties. We therefore hypothesized that 1,25-VitD3 promotes regeneration after vascular injury. METHODS AND RESULTS: In healthy volunteers, supplementation of vitamin D3 (4000 IU cholecalciferol per day) increased the number of circulating CD45-CD117+Sca1+Flk1+ angiogenic myeloid cells, which are thought to promote vascular regeneration. Similarly, in mice, 1,25-VitD3 (100 ng/kg per day) increased the number of angiogenic myeloid cells and promoted reendothelialization in the carotid artery injury model. In streptozotocin-induced diabetic mice, 1,25-VitD3 also promoted reendothelialization and restored the impaired angiogenesis in the femoral artery ligation model. Angiogenic myeloid cells home through the stromal cell-derived factor 1 (SDF1) receptor CXCR4. Inhibition of CXCR4 blocked 1,25-VitD3-stimulated healing, pointing to a role of SDF1. The combination of injury and 1,25-VitD3 increased SDF1 in vessels. Conditioned medium from injured, 1,25-VitD3-treated arteries elicited a chemotactic effect on angiogenic myeloid cells, which was blocked by SDF1-neutralizing antibodies. Conditional knockout of the vitamin D receptor in myeloid cells but not the endothelium or smooth muscle cells blocked the effects of 1,25-VitD3 on healing and prevented SDF1 formation. Mechanistically, 1,25-VitD3 increased hypoxia-inducible factor 1-α through binding to its promoter. Increased hypoxia-inducible factor signaling subsequently promoted SDF1 expression, as revealed by reporter assays and knockout and inhibitory strategies of hypoxia-inducible factor 1-α. CONCLUSIONS: By inducing SDF1, vitamin D3 is a novel approach to promote vascular repair.

Myeloid hypoxia-inducible factor-1α is essential for skeletal muscle regeneration in mice.

The outstanding regeneration ability of skeletal muscle is based on stem cells that become activated and develop to myoblasts after myotrauma. Proliferation and growth of myoblasts result in self-renewal of skeletal muscle. In this article, we show that myotrauma causes a hypoxic microenvironment leading to accumulation of the transcription factor hypoxia-inducible factor-1 (HIF-1) in skeletal muscle cells, as well as invading myeloid cells. To evaluate the impact of HIF-1 in skeletal muscle injury and repair, we examined mice with a conditional HIF-1α knockout targeted to skeletal muscle or myeloid cells in a model of soft tissue trauma. No differences in acute trauma size were detected between control and HIF-1α knockout mice. However, muscles of myeloid HIF-1α knockout mice showed a significant delay in myoblast proliferation and growth of regenerating myofibers, in association with decreased expression of cyclooxygenase-2 in HIF-1α-deficient myeloid cells. Moreover, the removal of necrotic cell debris and the regeneration of endothelial cell structure were impaired in myeloid HIF-1α knockout mice that showed delayed invasion of macrophages to the injury site. Our findings for the first time, to our knowledge, demonstrate that myeloid HIF-1α is required for adequate skeletal muscle regeneration.

Chronic hypoxia alters mitochondrial composition in human macrophages.

Hypoxia inducible factors (HIFs) are important mediators of the cellular adaptive response during acute hypoxia. The role of HIF-1 and HIF-2 during prolonged periods of hypoxia, i.e. chronic hypoxia is less defined. Therefore, we used human THP-1 macrophages with a knockdown of either HIF-1α, HIF-2α, or both HIFα-subunits, incubated them for several days under hypoxia (1% O2), and analyzed responses to hypoxia using 2D-DIGE coupled to MS/MS-analysis. Chronic hypoxia was defined as a time point when the early but transient accumulation of HIFα-subunits and mRNA expression of classical HIF target genes returned towards basal levels, with a new steady state that was constant from 72h onwards. From roughly 800 spots, that were regulated comparing normoxia to chronic hypoxia, about 100 proteins were unambiguously assigned during MS/MS-analysis. Interestingly, a number of glycolytic proteins were up-regulated, while a number of inner mitochondrial membrane proteins were down-regulated independently of HIF-1α or HIF-2α. Chronic hypoxic conditions depleted the mitochondrial mass by autophagy, which occurred independently of HIF proteins. Macrophages tolerate periods of chronic hypoxia very well and adaptive responses occur, at least in part, independently of HIF-1α and/or HIF-2α and comprise mitophagy as a pathway of particular importance.

HIF-1α is a negative regulator of plasmacytoid DC development in vitro and in vivo.

Hypoxia-inducible factors (HIFs) regulate hematopoiesis in the embryo and maintain hematopoietic stem cell function in the adult. How hypoxia and HIFs contribute to hematopoietic lineage differentiation in the adult is ill defined. Here we provide evidence that HIF-1 limits differentiation of precursors into plasmacytoid dendritic cells (pDCs). Low oxygen up-regulated inhibitor of DNA binding 2 (ID2) and suppressed Flt3-L-induced differentiation of bone marrow cells to pDCs in wild-type but not HIF-1α(fl/fl) LysM-Cre bone marrow cells. Moreover, pDC differentiated normally in hypoxic ID2(-/-) bone marrow cultures. Finally, we observed elevated pDC frequencies in bone marrow, blood, and spleen of HIF-1α(fl/fl) LysM-Cre and ID2(-/-), but not HIF-2α(fl/fl) LysM-Cre mice. Our data indicate that the low oxygen content in the bone marrow might limit pDC development. This might be an environmental mechanism to restrict the numbers of these potentially autoreactive cells.

Suppression of hypoxia-inducible factor-1α contributes to the antiangiogenic activity of red propolis polyphenols in human endothelial cells.

Polyphenol-enriched fractions from natural sources have been proposed to interfere with angiogenesis in pathological conditions. We recently reported that red propolis polyphenols (RPP) exert antiangiogenic activity. However, molecular mechanisms of this activity remain unclear. Here, we aimed at characterizing molecular mechanisms to explain the impact of RPP on endothelial cells' (EC) physiology. We used in vitro and ex and in vivo models to test the hypothesis that RPP inhibit angiogenesis by affecting hypoxia-inducible factor-1α (HIF1α) stabilization in EC. RPP (10 mg/L) affected angiogenesis by reducing migration and sprouting of EC, attenuated the formation of new blood vessels, and decreased the differentiation of embryonic stem cells into CD31-positive cells. Moreover, RPP (10 mg/L) inhibited hypoxia- or dimethyloxallylglycine-induced mRNA and protein expression of the crucial angiogenesis promoter vascular endothelial growth factor (VEGF) in a time-dependent manner. Under hypoxic conditions, RPP at 10 mg/L, supplied for 1-4 h, decreased HIF1α protein accumulation, which in turn attenuated VEGF gene expression. In addition, RPP reduced the HIF1α protein half-life from ~58 min to 38 min under hypoxic conditions. The reduced HIF1α protein half-life was associated with an increase in the von Hippel-Lindau (pVHL)-dependent proteasomal degradation of HIF1α. RPP (10 mg/L, 4 h) downregulated Cdc42 protein expression. This caused a corresponding increase in pVHL protein levels and a subsequent degradation of HIF1α. In summary, we have elucidated the underlying mechanism for the antiangiogenic action of RPP, which attenuates HIF1α protein accumulation and signaling.

Hypoxia inducible factor-dependent regulation of angiogenesis by nitro-fatty acids.

OBJECTIVE: Nitro-fatty acids (NO(2)-FAs) are emerging as a new class of cell signaling mediators. Because NO(2)-FAs are found in the vascular compartment and their impact on vascularization remains unknown, we aimed to investigate the role of NO(2)-FAs in angiogenesis. METHODS AND RESULTS: The effects of nitrolinoleic acid and nitrooleic acid were evaluated on migration of endothelial cell (EC) in vitro, EC sprouting ex vivo, and angiogenesis in the chorioallantoic membrane assay in vivo. At 10 µmol/L, both NO(2)-FAs induced EC migration and the formation of sprouts and promoted angiogenesis in vivo in an NO-dependent manner. In addition, NO(2)-FAs increased intracellular NO concentration, upregulated protein expression of the hypoxia inducible factor-1α (HIF-1α) transcription factor by an NO-mediated mechanism, and induced expression of HIF-1α target genes, such as vascular endothelial growth factor, glucose transporter-1, and adrenomedullin. Compared with typical NO donors such as spermine-NONOate and deta-NONOate, NO(2)-FAs were slightly less potent inducers of EC migration and HIF-1α expression. Short hairpin RNA-mediated knockdown of HIF-1α attenuated the induction of vascular endothelial growth factor mRNA expression and EC migration stimulated by NO(2)-FAs. CONCLUSION: Our data disclose a novel physiological role for NO(2)-FAs, indicating that these compounds induce angiogenesis in an NO-dependent mechanism via activation of HIF-1α.

Roles of hypoxia-inducible factor-1alpha (HIF-1alpha) versus HIF-2alpha in the survival of hepatocellular tumor spheroids.

UNLABELLED: 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-1alpha and HIF-2alpha in HepG2 cells and generated tumor spheroids as an in vitro hepatocellular carcinoma model. Knockdown of HIF-1alpha enhanced expression of HIF-2alpha and vice versa. Unexpectedly, knockdown of HIF-1alpha or HIF-2alpha increased cell viability as well as spheroid size and decreased caspase-3 activity. Antiapoptotic Bcl-X(L) expression increased in both knockdown spheroids, whereas proapoptotic Bax was only reduced in HIF-1alpha-knockdown cells. Furthermore, an HIF-2alpha-knockdown significantly increased Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) expression in an HIF-1alpha-dependent manner. Concomitantly, electron microscopy revealed a substantial increase in autophagosomal structures in HIF-2alpha-knockdown spheroids and mito-/lysotracker costaining confirmed lysosomal activity of these autophagosomes. Blocking autophagosome maturation using 3-methyladenine restored cell death in HIF-2alpha-knockdown clones comparable to wildtype cells. CONCLUSION: An HIF-1alpha-knockdown increases HIF-2alpha expression and shifts the balance of Bcl-2 family members toward survival. The knockdown of HIF-2alpha raises autophagic activity and attenuates apoptosis by enhancing HIF-1alpha expression. Our data indicate that enhanced expression of one HIF-isoform causes a survival advantage in hepatocellular carcinoma development.

The supernatant of apoptotic cells causes transcriptional activation of hypoxia-inducible factor-1alpha in macrophages via sphingosine-1-phosphate and transforming growth factor-beta.

Macrophages infiltrating solid tumors exhibit a tumor-supporting phenotype and are critical for tumor development. Little is known which tumor-derived signal provokes this phenotype shift and how these signals are interpreted in macrophages to support tumor growth. We used the supernatant of apoptotic cells and noticed transcriptional, nuclear factor of activated T cells-dependent up-regulation of hypoxia-inducible factor (HIF)-1alpha mRNA, subsequent protein expression, and HIF-1 activity. Blocking calcineurin with cyclosporine A attenuated nuclear factor of activated T cells binding during electrophoretic mobility shift assay analysis and circumvented the HIF-1alpha mRNA increase. Knockdown experiments, receptor analysis, and antibody neutralization pointed to sphingosine-1-phosphate and transforming growth factor-beta as the initiators of the HIF-1 response. The use of macrophages from conditional HIF-1alpha knockout mice revealed that macrophages, under the impact of apoptotic cell supernatants, use HIF-1 to produce factors that induce CD31 expression in murine embryonic stem cells. Our study supports the notion that soluble factors produced from apoptotic tumor cells activate the HIF-1 system under normoxia in macrophages to enhance their tumor-promoting capacity by, for example, releasing vascular endothelial growth factor. This shows the importance of HIF-1-elicited responses in regulatory macrophages under normoxia.

Hypoxia causes epigenetic gene regulation in macrophages by attenuating Jumonji histone demethylase activity.

Epigenetic processes elicit changes in gene expression by modifying DNA bases or histone side chains without altering DNA sequences. Recently discovered Jumonji histone demethylases (JHDMs) affect gene expression by demethylating lysine residues of histone tails. JHDMs belong to a family of dioxygenases and share similarities with prolyl hydroxylases (PHDs). Therefore, we investigated the influence of hypoxia in macrophages on histone methylation. All JHDM family members JMJD1A-C and JMJD2A-D are expressed in macrophages. Thus, we analyzed the methylation status of histone H3 residues not only under hypoxia but also after treatment with the dioxygenase-inhibitors DMOG, NO and ROS. Western analysis revealed increased methylations in H3K9me2/me3 and H3K36me3 at pO2 below 3%, DMOG, NO and ROS treatment. Chromatin immunoprecipitation (ChIP) assays demonstrated increased repressive marks H3K9me2 and H3K9me3 in specific promoter regions of the chemokine Ccl2 and the chemokine receptors Ccr1 and Ccr5, which correlated with a downregulation of their mRNA expression under hypoxic conditions. In contrasts, the hypoxia-inducible factor (HIF) target gene adrenomedullin (ADM) mRNA was upregulated and no increase in its histone modification was observed. We suggest that hypoxia and a concomitant loss of JHDM activity increases H3K9 methylation and decreases chemokine expression.