Hypoxia affects dendritic cell survival: role of the hypoxia-inducible factor-1α and lipopolysaccharide

Dendritic cells (DC) are the most potent antigen-presenting cells and during their life cycle they are exposed to different oxygen tensions. Similarly to inflamed and tumor tissues, lymphoid organs are characterized by a hypoxic microenvironment; thus, the modality by which hypoxia may affect DC is important for regulating both the quality and the intensity of the immune response. Here, we show that human monocyte-derived DC, exposed to hypoxia, expressed high levels of the hypoxia-inducible factor (HIF)-1α, associated with upregulation of BNIP3 and BAX expression. This was paralleled with downregulation of the anti-apoptotic molecule Bcl-2, enhanced caspase-3 activity and poly (ADP-ribose) polymerase cleavage, along with cell death. Transfection of HIF-1α siRNA protected DC from the effects of hypoxia. Of interest, when hypoxic DC were maturated with lipopolysaccharide (LPS), we did not observe an increased cell death, while HIF-1α accumulation and BNIP3 expression were still significantly upregulated. In contrast with immature DC, mature DC expressed higher levels of Bcl-2, and, more importantly, of phosphorylated Akt. Transfection of HIF-1α siRNA to mature DC resulted in a significant upregulation of Akt phosphorylation as well. Moreover, inhibition of PI3K/Akt pathway resulted in an increased cell death of hypoxic mature DC. We may conclude that a prolonged exposure to hypoxia induces a cell death program which could be prevented by HIF-1α inhibition and/or LPS maturation. Our results may contribute to further understand the physiology of DC and the molecular mechanisms involved in the survival of DC, with important implications in the regulation of the immune response.     

Oroxylin A increases the sensitivity of temozolomide on glioma cells by hypoxia-inducible factor 1α/hedgehog pathway under hypoxia

Microenvironmental hypoxia-mediated drug resistance is responsible for the failure of cancer therapy. To date, the role of the hedgehog pathway in resistance to temozolomide (TMZ) under hypoxia has not been investigated. In this study, we discovered that the increasing hypoxia-inducible factor 1α (HIF-1α) activated the hedgehog pathway in hypoxic microenvironment by promoting autocrine secretion of sonic hedgehog protein (Shh), and then upregulating transfer of Gli1 to the nucleus, finally contributed to TMZ resistance in glioma cells. Oroxylin A (C16H12O5), a bioactive flavonoid, could induce HIF-1α degradation via prolyl-hydroxylases–VHL signaling pathway, resulting in the inactivation of the hedgehog. Besides, oroxylin A increased the expression of Sufu, which is a negative regulator of Gli1. By this mechanism, oroxylin A sensitized TMZ on glioma cells. U251 intracranial transplantation model and GL261 xenograft model were used to confirm the reversal effects of oroxylin A in vivo. In conclusion, our results demonstrated that HIF-1α/hedgehog pathway conferred TMZ resistance under hypoxia, and oroxylin A was capable of increasing the sensitivity of TMZ on glioma cells in vitro and in vivo by inhibiting HIF-1α/hedgehog pathway and depressing the activation of Gli1 directly.     

Up-regulation of hypoxia-inducible factor-1α enhanced the cardioprotective effects of ischemic postconditioning in hyperlipidemic rats

Hyperlipidemia is an independent risk factor in the devel- opment of ischemic heart disease, which can increase myo- cardial susceptibility to ischemia/reperfusion （I/R） injury. Ischemic postconditioning （PostC） has now been demon- strated as a novel strategy to harness nature＇s protection against myocardial I/R injury in normal conditions. However, the effect of PostC on hyperlipidemic animals remains elusive. It has been shown in our previous study that PostC reduces the myocardial I/R injury, and hypoxia- inducible factor-1α （HIF-1α） may play an important role in the cardioprotective mechanisms of PostC on normal rats. Here, we tested the hypothesis that the cardioprotec- tion of PostC on hyperlipidemic rats is associated with the up-regulated HIF-1α expression. Male Wistar rats were fed with a high-fat diet for 8 weeks, and then randomly div- ided into five groups： sham, I/R, dimethyloxalylglycine （DMOG） ＋ I/R, PostC, and DMOG ＋ PostC group. The detrimental indices induced by I/R injury included infarct size, plasma creatine kinase （CK） activity and caspase-3 ac- tivity. The results showed that PostC could reduce the infarct size, when compared with the I/R group, which was consistent with the significant lower levels of plasma CK activity and caspase-3 activity, and that it increased the ex- pression of HIF-1α in hyperlipidemic rats. When DMOG was given before PostC to up-regulate HIF-1α protein level, the degree of I/R injury was attenuated. In conclu- sion, these data suggested that the up-regulation of HIF-1α may be one of the cardioprotective mechanisms of PostC against I/R injury in hyperlipidemic rats.     

Hypoxia-inducible factor-1α regulates the expression of L-type voltage-dependent Ca2+ channels in PC12 cells under hypoxia

Hypoxia is an important factor in regulation of cell behavior both under physiological and pathological conditions. The mechanisms of hypoxia-induced cell death have not been completely elucidated yet. It is well known that Ca²⁺ is critically related to cell survival. Hypoxia-inducible factor-1α (HIF-1α) is a core regulatory factor during hypoxia, and L-type voltage-dependent Ca²⁺ channels (L-VDCCs) have been reported to play a critical role in cell survival. This study was conducted to explore the relationship between L-VDCC expression and HIF-1α regulation in PC12 cells under hypoxia. PC12 cells were treated at 20 or 3 % O₂ to observe its proliferation and the intracellular calcium concentration. Then, we detected the protein expression of HIF-1α and L-VDCCs subtypes, Ca_v1.2 and Ca_v1.3. At last, to verify the relationship between HIF-1α and Ca_v1.2 and Ca_v1.3, we got the expression of Ca_v1.2 and Ca_v1.3 with Western blot and luciferase report gene assays after PC12 cells were treated by echinomycin, which is an HIF-1α inhibitor. Compared with 20 % O₂ (normoxia), 3 % O₂ (hypoxia) inhibited cell proliferation, increased the intracellular calcium concentration, and induced protein expression of HIF-1α. The protein expression of two L-VDCCs subtypes expressed in the nervous system, Ca_v1.2 and Ca_v1.3, was upregulated by hypoxia and reduced dose dependently by treatment with echinomycin, a HIF-1α inhibitor. Luciferase report gene assays showed that the expression of Ca_v1.2 and Ca_v1.3 genes was augmented under 3 % O₂. However, echinomycin only slightly and dose dependently decreased expression of the Ca_v1.2 gene, but not that of the Ca_v1.3 gene. These data indicated that Ca_v1.2 might be regulated by HIF-1α as one of its downstream target genes and involved in regulation of PC12 cells death under hypoxia.     

Effects of hypoxia and intracellular iron chelation on hypoxia-inducible factor-1α and -1β in the rat carotid body and glomus cells

The present investigation provides for the first time, unambiguous information on the occurrence of hypoxia-inducible factors (HIF-1 and HIF-1 proteins) in normoxia (Nx) and their interaction with hypoxia (Hx) and intracellular Fe²⁺ chelation in the rat carotid body (CB) glomus cells. HIF-1 bound to HIF-1 translocated into the nucleus is identified on the basis of immunohistochemistry and immunofluorescence. In Nx, a weak expression of HIF-1 was observed in CB glomus cells. However, exposure of CB and glomus cells to Hx (Po₂7 Torr) and Nx with ciclopirox olamine (CPX, 5 M) for 1 h showed a significant (P<0.001) increase in HIF-1 protein. The CBs and glomus cells exposed to Nx, Hx, and Nx with CPX showed a constant level of HIF-1 protein expression. HIF-1 subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. Hydroxylation of HIF-1 by prolyl hydroxylase for proteasomal degradation was dependent on iron, 2-oxoglutarate, and oxygen concentration. The intracellular iron that acts as a cofactor for prolyl hydroxylase activity belongs to the labile iron pool and can be easily chelated. Thus, chelation of intracellular labile iron by CPX in Nx significantly increased HIF-1 in CB glomus cells. Thus, the results are consistent with the hypothesis that HIF-1 which is present in the glomus cells translocates to the nucleus during exposure to Hx and to CPX in Nx.     

RETRACTED ARTICLE: Long noncoding RNA ANRIL is activated by hypoxia-inducible factor-1α and promotes osteosarcoma cell invasion and suppresses cell apoptosis upon hypoxia

Background

Osteosarcoma is the most common malignancy of bone. Intratumoral hypoxia occurs in many solid tumors, where it is associated with the development of aggressive phenotype. ANRIL has been shown to be a long noncoding RNA that facilitates the progression of a number of malignancies. Yet, few studies have explored the expression pattern of ANRIL in osteosarcoma and the effect of hypoxia on ANRIL.

Methods

We evaluated the expression levels of ANRIL in osteosarcoma tissues, adjacent normal tissues and cells with quantitative real-time polymerase chain reaction. Multiple approaches including luciferase reporter assay with nucleotide substitutions, chromatin immunoprecipitation assay and electrophoretic mobility shift assay were used to confirm the direct binding of HIF-1α to the ANRIL promoter region. SiRNA-based knockdown and other molecular biology techniques were employed to measure the effect of HIF-1α on the expression of ANRIL.

Results

We found that the expression of ANRIL was upregulated in 15 pairs of osteosarcoma compared with adjacent normal tissues. We found that hypoxia is sufficient to upregulate ANRIL expression in osteosarcoma cells (MNNG and U2OS). HIF-1α directly binds to the putative hypoxia response element in the upstream region of ANRIL. What’s more, siRNA and small molecular inhibitors-mediated HIF-1α suppression attenuated ANRIL upregulation under hypoxic conditions. Upon hypoxia, ANRIL promoted cancer cell invasion and suppressed cell apoptosis.

Conclusion

Taken together, these data suggest that HIF-1α may contribute to the upregulation of ANRIL in osteosarcoma under hypoxic conditions. ANRIL is involved in hypoxia-induced aggressive phenotype in osteosarcoma.

     

On the value of Elongation factor-1α for reconstructing pterygote insect phylogeny

Pterygota are traditionally divided in two lineages, the “Palaeoptera” and Neoptera. Despite several efforts neither morphology nor molecular systematics have resolved the phylogeny of the pterygote insects. Too few markers have yet been identified for adequately tracking mesozoic-aged divergences. We tested the Elongation factor-1α for its phylogenetic value in pterygote insect systematics. This highly conserved nuclear protein-coding gene has previously been reported to be useful in other groups for phylogenetic analyses at the intraordinal level as well as at the interordinal level. The analyses suggest that EF-1α DNA sequences as well as intron positions provide informative markers for pterygote phylogenetics.     

Calcineurin B subunit interacts with proteasome subunit alpha type 7 and represses hypoxia-inducible factor-1α activity via the proteasome pathway

The calcineurin (CN) B subunit (CNB) is the regulatory subunit of CN, which is the only serine/threonine-specific protein phosphatase regulated by Ca²⁺/CaM. It has been shown to have potential as an anticancer agent, and has a positive effect on the phagocytic index and coefficient. We report here that CNB binds to proteasome subunit alpha type 7 (PSMA7) and inhibits the transactivation activity of hypoxia-inducible factor-1α (HIF-1α) via the proteasome pathway. In addition, we show that CNB represses the expression of vascular endothelial growth factor (VEGF), which is regulated by HIF-1α. These results indicate that CNB modulates cellular proteasome activity via a specific interaction with PSMA7. This may provide a molecular basis for its anticancer and antiviral activities.