HIF-1α Abundance Research Articles

STAT3 palmitoylation initiates a positive feedback loop that promotes the malignancy of hepatocellular carcinoma cells in mice.

Constitutive activation of the transcription factor STAT3 (signal transducer and activator of transcription 3) contributes to the malignancy of many cancers such as hepatocellular carcinoma (HCC) and is associated with poor prognosis. STAT3 activity is increased by the reversible palmitoylation of Cys108 by the palmitoyltransferase DHHC7 (encoded by ZDHHC7). Here, we investigated the consequences of S-palmitoylation of STAT3 in HCC. Increased ZDHHC7 abundance in HCC cases was associated with poor prognosis, as revealed by bioinformatics analysis of patient data. In HepG2 cells in vitro, DHHC7-mediated palmitoylation enhanced the expression of STAT3 target genes, including HIF1A, which encodes the hypoxia-inducible transcription factor HIF1α. Inhibiting DHHC7 decreased the S-palmitoylation of STAT3 and decreased HIF1α abundance. Furthermore, stabilization of HIF1α by cyclin-dependent kinase 5 (CDK5) enabled it to promote the expression of ZDHHC7, which generated a positive feedback loop between DHHC7, STAT3, and HIF1α. Perturbing this loop reduced the growth of HCC cells in vivo. Moreover, DHHC7, STAT3, and HIF1α were all abundant in human HCC tissues. Our study identifies a pathway connecting these proteins that is initiated by S-palmitoylation, which may be broadly applicable to understanding the role of this modification in cancer.

Canagliflozin alleviates high glucose-induced peritoneal fibrosis via HIF-1α inhibition.

The cardioprotective effects of sodium-glucose cotransporter type 2 (SGLT2) inhibitors have been demonstrated in many studies. However, their benefits for end-stage kidney disease patients, particularly those on peritoneal dialysis, remain unclear. SGLT2 inhibition has shown peritoneal protective effects in some studies, but the mechanisms are still unknown. Herein, we investigated the peritoneal protective mechanisms of Canagliflozin in vitro by simulating hypoxia with CoCl2 in human peritoneal mesothelial cells (HPMCs) and rats by intraperitoneal injection of 4.25% peritoneal dialysate simulating chronic high glucose exposure. CoCl2 hypoxic intervention significantly increased HIF-1α abundance in HPMCs, activated TGF-β/p-Smad3 signaling, and promoted the production of fibrotic proteins (Fibronectin, COL1A2, and α-SMA). Meanwhile, Canagliflozin significantly improved the hypoxia of HPMCs, decreased HIF-1α abundance, inhibited TGF-β/p-Smad3 signaling, and decreased the expression of fibrotic proteins. Five-week intraperitoneal injection of 4.25% peritoneal dialysate remarkably increased peritoneal HIF-1α/TGF-β/p-Smad3 signaling and promoted peritoneal fibrosis and peritoneal thickening. At the same time, Canagliflozin significantly inhibited the HIF-1α/TGF-β/p-Smad3 signaling, prevented peritoneal fibrosis and peritoneal thickening, and improved peritoneal transportation and ultrafiltration. High glucose peritoneal dialysate increased the expression of peritoneal GLUT1, GLUT3 and SGLT2, all of which were inhibited by Canagliflozin. In conclusion, we showed that Canagliflozin could improve peritoneal fibrosis and function by ameliorating peritoneal hypoxia and inhibiting the HIF-1α/TGF-β/p-Smad3 signaling pathway, providing theoretical support for the clinical use of SGLT2 inhibitors in patients on peritoneal dialysis.

A novel AMPK activator shows therapeutic potential in hepatocellular carcinoma by suppressing HIF1α-mediated aerobic glycolysis.

Hepatocellular carcinoma (HCC) is characterized by rapid growth, early vascular invasion, and high metastasis. Currently available US Food and Drug Administration (FDA)‐approved drugs show low therapeutic efficacy, limiting HCC treatment to chemotherapy. We designed and synthesized a novel small molecule, SCT‐1015, that allosterically activated adenosine monophosphate‐activated protein kinase (AMPK) to suppress the aerobic glycolysis in HCC. SCT‐1015 was shown to bind the AMPK α and β‐subunit interface, thereby exposing the kinase α domain to the upstream kinases, resulting in the increased AMPK activity. SCT‐1015 dramatically reduced HCC cell growth in vitro and tumor growth in vivo. We further found that AMPK formed protein complexes with hypoxia‐inducible factor 1‐alpha (HIF1α) and that SCT‐1015‐activated AMPK promoted hydroxylation of HIF1α (402P and 564P), resulting in HIF1α degradation by the ubiquitin‐proteasome system. With declined HIF1α abundance, many glycolysis‐related enzymes were downregulated, suppressing aerobic glycolysis, and promoting oxidative phosphorylation. These results indicated that SCT‐1015 channeled HCC cells into an unfavorable metabolic status. Overall, we reported SCT‐1015 as a direct activator of AMPK signaling that held therapeutic potential in HCC.

Phosphodiesterase 10A Is a Critical Target for Neuroprotection in a Mouse Model of Ischemic Stroke.

Phosphodiesterase 10A (PDE10A) hydrolyzes adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP). It is highly expressed in the striatum. Recent evidence implied that PDE10A may be involved in the inflammatory processes following injury, such as ischemic stroke. Its role in ischemic injury was unknown. Herein, we exposed mice to 90 or 30-min middle cerebral artery occlusion, followed by the delivery of the highly selective PDE10A inhibitor TAK-063 (0.3mg/kg or 3mg/kg) immediately after reperfusion. Animals were sacrificed after 24 or 72h, respectively. Both TAK-063 doses enhanced neurological function, reduced infarct volume, increased neuronal survival, reduced brain edema, and increased blood-brain barrier integrity, alongside cerebral microcirculation improvements. Post-ischemic neuroprotection was associated with increased phosphorylation (i.e., activation) of pro-survival Akt, Erk-1/2, GSK-3α/βand anti-apoptotic Bcl-xL abundance, decreased phosphorylation of pro-survival mTOR, and HIF-1α, MMP-9 and pro-apoptotic Bax abundance. Interestingly, PDE10A inhibition reduced inflammatory cytokines/chemokines, including IFN-γ and TNF-α, analyzed by planar surface immunoassay. In addition, liquid chromatography-tandem mass spectrometry revealed 40 proteins were significantly altered by TAK-063. Our study established PDE10A as a target for ischemic stroke therapy.

Hypoxia‐Induced TIMAP Upregulation Promotes Tumor Angiogenesis by Attenuating ALK1‐Mediated TIMAP Repression

Introduction TIMAP (TGF-β1 inhibited membrane associated protein) is an endothelial cell (EC)-predominant inhibitory protein phosphatase 1β (PP1β) regulatory subunit in the myosin phosphatase targeting (MYPT) family (J. Biol. Chem. 2294:13280, 2019). TIMAP promotes proliferation and survival of EC and enhances in vitro sprouting angiogenesis (Am. J. Physiol. - Renal Physiol. 307: F623, 2014). Hypothesis Hypoxia stimulates TIMAP expression in EC, enhancing in vivo tumor angiogenesis. Methods Mammary adenocarcinoma cells (E0771, syngeneic for C57BL/6 mice) were injected into the mammary glands of five pairs of female TIMAP+/+ / TIMAP-/- C57BL/6 mice at 6-8 weeks of age. For each pair, mice were euthanized on the day the tumor diameter exceeded 1.5 cm in one mouse. Tumor weight, mean tumor diameter, and tumor vascular density were then quantified. For quantification of vascular density, the EC markers were PECAM1 and Tie-2 for fluorescence microscopy (IF) and western blot (WB) analysis, respectively. TIMAP+/+ mice were also exposed to progressive, chronic hypoxia by reducing the ambient O2 concentration from 18 to 10 % over two weeks and then maintaining it at 10% for 3 weeks. Lung tissue was then evaluated for TIMAP abundance by WB analysis, and TIMAP localization was determined by IF. Finally, the mechanism regulating TIMAP expression was evaluated in cultured EC in the presence of 21% and 1% O2 with or without TGF-β pathway activation or inhibition. Results Tumor size was similar in TIMAP+/+ and TIMAP-/- mice through days 8-10 after injection of E0771 cells. In 4 of 5 TIMAP-/-, but not TIMAP+/+ mice, the tumors then eroded partially through the skin and regressed gradually. At the time of euthanasia tumor size (Fig. 1A), weight (Fig. 1B) and blood vessel density (Fig.1C, D&E) were significantly lower in TIMAP-/- compared to TIMAP+/+ mice. In the chronic hypoxia model, TIMAP protein abundance in lung lysates was significantly higher in hypoxic compared to control mice, and IF using anti-TIMAP and anti-PECAM1 antibodies revealed that the TIMAP protein was restricted to lung EC. In cultured EC, HIF-1α and TIMAP protein abundance were significantly higher in EC exposed to 1% than 21% O2, (Fig. 1F,G). The effect of 1% O2 on TIMAP was only observed in the presence of fresh 1% serum or BMP-9 (1-10 ng/ml), while the hypoxia-induced increase in HIF-1α abundance was independent of serum or BMP-9. In the absence of hypoxia 1% serum and BMP-9 strongly stimulated Smad 1/5/8 but not Smad 2/3 phosphorylation and significantly reduced TIMAP protein levels. The ALK1 inhibitor K02288 but not the ALK 5 inhibitor LY-364947 blocked 1% serum and BMP-9 stimulated Smad 1/5/8 phosphorylation and raised the EC TIMAP abundance. Notably, hypoxia mimicked the ALK1 inhibitor, significantly blunting 1% serum and BMP-9-stimulated Smad 1/5/8 phosphorylation. Conclusion Hence, hypoxia induces expression of the pro-angiogenic PP1β inhibitor TIMAP due, at least in part, to inhibition of BMP-9/ALK1-mediated repression of TIMAP. Furthermore, TIMAP is necessary for effective tumor angiogenesis in vivo. We therefore speculate that tumor hypoxia stimulates EC TIMAP expression by inhibiting the ALK1 pathway.

Intrabody against prolyl hydroxylase 2 ameliorates acetaminophen-induced acute liver injury in mice via concomitant promotion of angiogenesis and redox homeostasis

Acetaminophen (APAP) overdose has become the most common cause of drug-induced acute liver failure. Angiogenesis and redox homeostasis play an important role in liver protection and repair of APAP-induced acute liver injury (AILI). Hypoxia inducible factor-1 (HIF-1) is a transcription factor that plays a crucial role in regulating the expression of genes associated with angiogenesis, redox homeostasis and energy balance. Prolyl hydroxylase 2 (PHD2) predominantly hydroxylates proline residues in HIF-1α to promote its degradation. In our previous study, we reported an intrabody against PHD2 (ER-INP) that enhances angiogenesis by blocking PHD2 activity to increase HIF-1α abundance and activity. The present study was designed to explore the role and possible mechanisms of ER-INP in AILI in mice. Mice were pretreated intravenously with ER-INP before intraperitoneal injection of APAP to induce AILI. The results showed that pretreatment with ER-INP dramatically decreased the high ALT and AST activities and significantly ameliorated the centrilobular necrosis induced by APAP administration. ER-INP expression promoted angiogenesis in vivo by upregulating the mRNA and protein levels of HIF-1α target genes. Meanwhile, ER-INP pretreatment restored redox homeostasis, verified by reinforcement of PRDX4 activity and suppression of GSH depletion. This study demonstrated that ER-INP protects against AILI in part by increasing angiogenesis and maintaining redox homeostasis. These results indicate that ER-INP may provide a potential liver protection strategy against AILI in the future.

Intrabody against prolyl hydroxylase 2 promotes angiogenesis by stabilizing hypoxia-inducible factor-1\u03b1

Hypoxia-inducible factor (HIF)-1α is a crucial transcription factor that regulates the expression of target genes involved in angiogenesis. Prolyl hydroxylase 2 (PHD2) dominantly hydroxylates two highly conserved proline residues of HIF-1α to promote its degradation. This study was designed to construct an intrabody against PHD2 that can inhibit PHD2 activity and promote angiogenesis. Single-chain variable fragment (scFv) against PHD2, INP, was isolated by phage display technique and was modified with an endoplasmic reticulum (ER) sequence to obtain ER-retained intrabody against PHD2 (ER-INP). ER-INP was efficiently expressed and bound to PHD2 in cells, significantly increased the levels of HIF-1α, and decreased hydroxylated HIF-1α in human embryonic kidney cell line (HEK293) cells and mouse mononuclear macrophage leukaemia cell line (RAW264.7) cells. ER-INP has shown distinct angiogenic activity both in vitro and in vivo, as ER-INP expression significantly promoted the migration and tube formation of human umbilical vein endothelial cells (HUVECs) and enhanced angiogenesis of chick chorioallantoic membranes (CAMs). Furthermore, ER-INP promoted distinct expression and secretion of a range of angiogenic factors. To the best of our knowledge, this is the first study to report an ER-INP intrabody enhancing angiogenesis by blocking PHD2 activity to increase HIF-1α abundance and activity. These results indicate that ER-INP may play a role in the clinical treatment of tissue injury and ischemic diseases in the future.

Lactate-Dependent Chaperone-Mediated Autophagy Induces Oscillatory Hif-1α Activity Promoting Proliferation of Hypoxic Cells

Response to hypoxia is a highly regulated process controlling multiple cell and tissue level functions. However, we still know little about dynamic single cell responses to hypoxic conditions. Here, using fluorescent reporters of hypoxia response factor-1α (HIF-1α) activity, we show that hypoxic responses in individual cells can be highly dynamic and variable across the population. These responses fall into three classes, including oscillatory activity. We identify a molecular mechanism that can account for all three response classes, demonstrating that the oscillations of HIF-1α activity and abundance are controlled by the reactive oxygen species-dependent chaperone-mediated autophagy in a respiring subset of cells. Furthermore, we find that the oscillatory response is modulated by the abundance of extracellular lactate in a quorum sensing-like mechanism. We show that oscillatory HIF-1α activity can help rescue hypoxia-mediated inhibition of cell division, suggesting a mechanism for aggressive behavior in a subset of hypoxic tumor cells.

Regulation of Hypoxia-Inducible Factor 1α during Hypoxia by DAP5-Induced Translation of PHD2.

Death-associated protein 5 (DAP5) is an atypical isoform of the translation initiation scaffolds eukaryotic initiation factor 4GI (eIF4GI) and eIF4GII (eIF4GI/II), which recruit mRNAs to ribosomes in mammals. Unlike eIF4GI/II, DAP5 binds eIF2β, a subunit of the eIF2 complex that delivers methionyl-tRNA to ribosomes. We discovered that DAP5:eIF2β binding depends on specific stimuli, e.g., protein kinase C (PKC)-Raf-extracellular signal-regulated kinase 1/2 (ERK1/2) signals, and determines DAP5's influence on global and template-specific translation. DAP5 depletion caused an unanticipated surge of hypoxia-inducible factor 1α (HIF-1α), the transcription factor and master switch of the hypoxia response. Physiologically, the hypoxia response is tempered through HIF-1α hydroxylation by the oxygen-sensing prolyl hydroxylase-domain protein 2 (PHD2) and subsequent ubiquitination and degradation. We found that DAP5 regulates HIF-1α abundance through DAP5:eIF2β-dependent translation of PHD2. DAP5:eIF2-induced PHD2 translation occurred during hypoxia-associated protein synthesis repression, indicating a role as a safeguard to reverse HIF-1α accumulation and curb the hypoxic response.

Abstract P419: Klotho Ameliorate Medullary Fibrosis and Pressure Natriuresis in Hypertensive Rat Kidneys

Recent data indicate that klotho interacts with the renin-angiotensin system (RAS). However, the effects of klotho protein supplementation on hypertensive renal injury have not been examined yet. First, the in vivo experiments were performed using spontaneously hypertensive rats (SHR). To elucidate in vivo observations, the in vitro studies were carried out to examine the interactions between klotho protein and angiotensin type-1 (AT1) receptor with immunoprecipitation and cell culture (HK-2 cell) methods. Uninephrectomized SHRs were treated with exogenous klotho protein or vehicle. Exogenous klotho protein supplementation to uninephrectomized SHRs decreased blood pressure, renal angiotensin II levels, angiotensinogen expression, HIF-1α abundance, and medullary fibronectin with increased renal klotho expression and serum and urine klotho levels. Klotho supplementation also reduced kidney weight, renal phosphorylated Akt and mTOR abundance. Furthermore, klotho supplementation restored renal autoregulation of glomerular filtration rate and renal plasma flow, and improved pressure-induced natriuresis in SHR. Klotho protein bound to AT1 receptors without affecting the bindings of angiotensin II to AT1 receptor. Klotho decreased the presence of AT1 receptors on HK-2 cells, attenuating inositol triphosphate generation. Angiotensin II elevated angiotensinogen expression in HK-2 cells, and klotho protein suppressed angiotensin II-induced increases in angiotensinogen expression. Of note, although angiotensin II improves glomerular autoregulatory ability especially at lower pressure range, angiotensin II impairs pressure-induced natriuresis. Collectively, the present data demonstrate that klotho binds with the AT1 receptors to suppress angiotensin signal transduction and inactivate the renal RAS. In addition, our results suggest that exogenous klotho supplementation suppresses Akt-mTOR signaling to decline renal hypertrophy and restores renal autoregulatory ability in uninephrectomized SHRs. Finally, the present findings implicate that klotho supplementation inhibits HIF-1α pathway to suppress medullary fibrosis, presumably involved in improvements of pressure-natriuresis and reduction in blood pressure.

A stiff path to aggressiveness

Whether a glioblastoma-associated IDH1 mutant can prevent the hypoxic induction of HIF1α depends on the stiffness of the extracellular matrix.

Attenuated PGC-1α Isoforms following Endurance Exercise with Blood Flow Restriction.

Exercise performed with blood flow restriction simultaneously enhances the acute responses to both myogenic and mitochondrial pathways with roles in training adaptation. We investigated isoform-specific gene expression of the peroxisome proliferator-activated receptor gamma coactivator 1 and selected target genes and proteins regulating skeletal muscle training adaptation. Nine healthy, untrained males participated in a randomized, counterbalanced, crossover design in which each subject completed a bout of low-intensity endurance exercise performed with blood flow restriction (15 min cycling at 40% of V˙O2peak, BFR-EE), endurance exercise (30 min cycling at 70% of V˙O2peak, EE), or resistance exercise (4 × 10 repetitions of leg press at 70% of one-repetition maximum) separated by at least 1 wk of recovery. A single resting muscle biopsy (vastus lateralis) was obtained 2 wk before the first exercise trial (rest) and 3 h after each bout. Total PGC-1α mRNA abundance, along with all four isoforms, increased above rest with EE only (P < 0.05) being higher than BFR-EE (P < 0.05). PGC-1α1, 2, and 4 were higher after EE compared with resistance exercise (P < 0.05). EE also increased vascular endothelial growth factor, Hif-1α, and MuRF-1 mRNA abundance above rest (P < 0.05), whereas COXIV mRNA expression increased with EE compared with BFR-EE (P < 0.05). The attenuated expression of all four PGC-1α isoforms when EE is performed with blood flow restriction suggests this type of exercise provides an insufficient stimulus to activate the signaling pathways governing mitochondrial and angiogenesis responses observed with moderate- to high-intensity EE.

Radiation Promptly Alters Cancer Live Cell Metabolic Fluxes: An In Vitro Demonstration.

Quantitative data is presented that shows significant changes in cellular metabolism in a head and neck cancer cell line 30 min after irradiation. A head and neck cancer cell line (UM-SCC-22B) and a comparable normal cell line, normal oral keratinocyte (NOK) were each separately exposed to 10 Gy and treated with a control drug for disrupting metabolism (potassium cyanide; KCN). The metabolic changes were measured live by fluorescence lifetime imaging of the intrinsically fluorescent intermediate metabolite nicotinamide adenosine dinucleotide (NADH) fluorescence; this method is sensitive to the ratio of bound to free NADH. The results indicated a prompt shift in metabolic signature in the cancer cell line, but not in the normal cell line. Control KCN treatment demonstrated expected metabolic fluxes due to mitochondrial disruption. The detected radiation shift in the cancer cells was blunted in the presence of both a radical scavenger and a HIF-1α inhibitor. The HIF-1α abundance as detected by immunohistochemical staining also increased substantially for these cancer cells, but not for the normal cells. This type of live-cell metabolic monitoring could be helpful for future real-time studies and in designing adaptive radiotherapy approaches.

HIF-1-alpha links mitochondrial perturbation to the dynamic acquisition of breast cancer tumorigenicity.

Up-regulation of hypoxia-inducible factor-1α (HIF-1α), even in normoxia, is a common feature of solid malignancies. However, the mechanisms of increased HIF-1α abundance, and its role in regulating breast cancer plasticity are not fully understood. We have previously demonstrated that dimethyl-2-ketoglutarate (DKG), a widely used cell membrane-permeable α-ketoglutarate (α-KG) analogue, transiently stabilizes HIF-1α by inhibiting prolyl hydroxylase 2. Here, we report that breast cancer tumorigenicity can be acquired through prolonged treatment with DKG. Our results indicate that, in response to prolonged DKG treatment, mitochondrial respiration becomes uncoupled, leading to the accumulation of succinate and fumarate in breast cancer cells. Further, we found that an early increase in the oxygen flux rate was accompanied by a delayed enhancement of glycolysis. Together, our results indicate that these events trigger a dynamic enrichment for cells with pluripotent/stem-like cell markers and tumorsphere-forming capacity. Moreover, DKG-mediated metabolic reprogramming results in HIF-1α induction and reductive carboxylation pathway activation. Both HIF-1α accumulation and the tumor-promoting metabolic state are required for DKG-promoted tumor repopulation capacity in vivo. Our data suggest that mitochondrial adaptation to DKG elevates the ratio of succinate or fumarate to α-KG, which in turn stabilizes HIF-1α and reprograms breast cancer cells into a stem-like state. Therefore, our results demonstrate that metabolic regulation, with succinate and/or fumarate accumulation, governs the dynamic transition of breast cancer tumorigenic states and we suggest that HIF-1α is indispensable for breast cancer tumorigenicity.

HSP90 inhibits apoptosis and promotes growth by regulating HIF-1α abundance in hepatocellular carcinoma.

Heat shock protein (HSP)90 functions as a general oncogene by targeting several well-known oncoproteins for ubiquination and proteasomal degradation. However, the clinical significance of HSP90, as well as the mechanisms responsible for the tumor-promoting effects of HSP90 in hepatocellular carcinoma (HCC) remain unclear. In this study, HSP90 and hypoxia-inducible factor (HIF)-1α expression in 60 samples of HCC tissues and matched normal tumor-adjacent tissue were assessed using immunohistochemistry (IHC) or western blot analysis. Flow cytometry, BrdU cell proliferation assay, caspase-3/7 activity assay and MTT assay were used to detect the apoptosis and proliferation of the HCC cells. The regulatory effect of HSP90 on HIF-1α in the HCC cells was confirmed by immunofluorescence staining, western blot analysis and RT-qPCR. The interaction between HIF-1α and HSP90 was analyzed by co-immunoprecipitation. A subcutaneous tumor xenograft model in nude mice was established and TUNEL assay was performed to evaluate cancer cell apoptosis and growth in vivo. We found that HSP90 expression was higher in the HCC tissues than in the normal tissues and that a high HSP90 expression correlated with poor clinicopathological characteristics, including venous infiltration, an advanced TNM stage and high pathological grading. Furthermore, we confirmed that patients with a negative expression of HSP90 had an improved 3-year survival, and that HSP90 was an independent factor for predicting the prognosis of patients with HCC. We demonstrated that HSP90 promoted HCC by inhibiting apoptosis and promoting cancer cell growth. Pearson's correlation coefficient analysis indicated that HSP90 expression positively correlated with HIF-1α protein expression in the HCC tissues. Furthermore, we found that HSP90 regulated HIF-1α protein abundance by inhibiting the ubiquitination and proteasomal degradation of HIF-1α in HCC cells. Additionally, the upregulation of HIF-1α expression partially abrogated HSP90 siRNA-induced HCC cell growth arrest and apoptosis in vitro and in vivo. These results indicate that HSP90 may be used as a prognostic marker and that HIF-1α may be one of the potential therapeutic targets of HSP90 in HCC.

Systemic Proteasome Inhibition Induces Sustained Post-stroke Neurological Recovery and Neuroprotection via Mechanisms Involving Reversal of Peripheral Immunosuppression and Preservation of Blood-Brain-Barrier Integrity.

In view of its profound effect on cell survival and function, the modulation of the ubiquitin-proteasome-system has recently been shown to promote neurological recovery and brain remodeling after focal cerebral ischemia. Hitherto, local intracerebral delivery strategies were used, which can hardly be translated to human patients. We herein analyzed effects of systemic intraperitoneal delivery of the proteasome inhibitor BSc2118 on neurological recovery, brain injury, peripheral and cerebral immune responses, neurovascular integrity, as well as cerebral neurogenesis and angiogenesis in a mouse model of transient intraluminal middle cerebral artery occlusion. Systemic delivery of BSc2118 induced acute neuroprotection reflected by reduced infarct volume when delivered up to 9h post-stroke. The latter was associated with reduced brain edema and stabilization of blood-brain-barrier integrity, albeit cerebral proteasome activity was only mildly reduced. Neuronal survival persisted in the post-acute stroke phase up to 28days post-stroke and was associated with improved neurological recovery when the proteasome inhibitor was continuously delivered over 7days. Systemic proteasome inhibition prevented stroke-induced acute leukocytosis in peripheral blood and reversed the subsequent immunosuppression, namely, the reduction of blood lymphocyte and granulocyte counts. On the contrary, post-ischemic brain inflammation, cerebral HIF-1α abundance, cell proliferation, neurogenesis, and angiogenesis were not influenced by the proteasome inhibitor. The modulation of peripheral immune responses might thus represent an attractive target for the clinical translation of proteasome inhibitors.

Intermediary metabolite precursor dimethyl-2-ketoglutarate stabilizes hypoxia-inducible factor-1α by inhibiting prolyl-4-hydroxylase PHD2.

Hypoxia-inducible factor 1α (HIF-1α), a major mediator of tumor physiology, is activated during tumor progression, and its abundance is correlated with therapeutic resistance in a broad range of solid tumors. The accumulation of HIF-1α is mainly caused by hypoxia or through the mutated succinate dehydrogenase A (SDHA) or fumarate hydratase (FH) expression to inhibit its degradation. However, its activation under normoxic conditions, termed pseudohypoxia, in cells without mutated SDHA or FH is not well documented. Here, we show that dimethyl-2-ketoglutarate (DKG), a cell membrane-permeable precursor of a key metabolic intermediate, α-ketoglutarate (α-KG), known for its ability to rescue glutamine deficiency, transiently stabilized HIF-1α by inhibiting activity of the HIF prolyl hydroxylase domain-containing protein, PHD2. Consequently, prolonged DKG-treatment under normoxia elevated HIF-1α abundance and up-regulated the expression of its downstream target genes, thereby inducing a pseudohypoxic condition. This HIF-1α stabilization phenotype is similar to that from treatment of cells with desferrioxamine (DFO), an iron chelator, or dimethyloxalyglycine (DMOG), an established PHD inhibitor, but was not recapitulated with other α-KG analogues, such as Octyl-2KG, MPTOM001 and MPTOM002. Our study is the first example of an α-KG precursor to increase HIF-1α abundance and activity. We propose that DKG acts as a potent HIF-1α activator, highlighting the potential use of DKG to investigate the contribution of PHD2-HIF-1α pathway to tumor biology.

Negative regulation of Hif1a expression and TH17 differentiation by the hypoxia-regulated microRNA miR-210.

MicroRNA-210 (miR-210) is a signature microRNA of hypoxia. We found robust increase (>100-fold) of miR-210 abundance in activated T cells, especially in the TH17 lineage. Hypoxia synergized with T cell receptor (TCR)–CD28 stimulation to accelerate and increase the magnitude of Mir210 expression. Mir210 was directly regulated by HIF-1α, a key regulator of TH17 polarization. Surprisingly, Hif1a was identified as a miR-210-target, suggesting negative-feedback by miR-210 to inhibit HIF-1α protein expression. Deletion of Mir210 promoted TH17 differentiation under conditions with limited oxygen. In experimental colitis, miR-210 reduced Hif1a transcript abundance, reduced the proportion of cells producing inflammatory cytokines and controlled disease severity. Our study identifies miR-210 as an important regulator of T cell differentiation in hypoxia, which can limit immunopathology.

Another road leads to HIF-1 activation: implications for prostate cancer progression

Another road leads to HIF-1 activation: implications for prostate cancer progression

The ATP-binding cassette transporters ABCB1 and ABCC1 are not regulated by hypoxia in immortalised human brain microvascular endothelial cells

BackgroundATP-binding cassette transporters at the blood-brain barrier are actively regulated upon ischemic stroke in a way that impedes the access of pharmacological compounds to the brain tissue. The luminal endothelial transporter ABCB1 was recently shown to be increased, whereas the abluminal transporter ABCC1 was decreased on ischemic brain capillaries. In vitro studies using epithelial cells suggested that ABCB1 is regulated during hypoxia in a hypoxia-inducible factor (HIF)-1α-dependent way.MethodsIn order to investigate whether hypoxia might be responsible for the expression changes of ABCB1 and ABCC1 in the ischemic brain, the immortalised human brain microvascular endothelial cell line hCMEC/D3 was exposed to hypoxia (1%) or anoxia (0%). Cell lysates were analysed by Western blot to detect the protein expression of ABCB1, ABCC1, HIF-1α and HIF-2α.ResultsDuring hypoxia, an accumulation of HIF-1α and HIF-2α was noticed in hCMEC/D3 cells that followed different time kinetics. Both HIF-1α and HIF-2α abundance increased within 4 h of hypoxia. HIF-1α levels decreased to below detection levels within 16 h of hypoxia, whereas HIF-2α remained elevated even after 48 h. No changes of ABCB1 and ABCC1 expression were detected, neither on the mRNA nor protein level.ConclusionOur data suggests that other factors than hypoxia may be responsible for the expression changes of ATP-binding cassette transporters in the ischemic brain.