ERRα augments HIF‐1 signalling by directly interacting with HIF‐1α in normoxic and hypoxic prostate cancer cells

Adaptation of cancer cells to a hypoxic microenvironment is important for their facilitated malignant growth and advanced development. One major mechanism mediating the hypoxic response involves up‐regulation of hypoxia‐inducible factor 1 (HIF‐1) expression, which controls reprogramming of energy metabolism and angiogenesis. Oestrogen‐related receptor‐α (ERRα) is a pivotal regulator of cellular energy metabolism and many biosynthetic pathways, and has also been proposed to be an important factor promoting the Warburg effect in advanced cancer. We and others have previously shown that ERRα expression is increased in prostate cancer and is also a prognostic marker. Here we show that ERRα is oncogenic in prostate cancer and also a key hypoxic growth regulator. ERRα‐over‐expressing prostate cancer cells were more resistant to hypoxia and showed enhanced HIF‐1α protein expression and HIF‐1 signalling. These effects could also be observed in ERRα‐over‐expressing cells grown under normoxia, suggesting that ERRα could function to pre‐adapt cancer cells to meet hypoxia stress. Immunoprecipitation and FRET assays indicated that ERRα could physically interact with HIF‐1α via its AF‐2 domain. A ubiquitination assay showed that this ERRα–HIF‐1α interaction could inhibit ubiquitination of HIF‐1α and thus reduce its degradation. Such ERRα–HIF‐1α interaction could be attenuated by XCT790, an ERRα‐specific inverse agonist, resulting in reduced HIF‐1α levels. In summary, we show that ERRα can promote the hypoxic growth adaptation of prostate cancer cells via a protective interaction with HIF‐1α, suggesting ERRα as a potential therapeutic target for cancer treatment. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Vitexin alleviates interleukin‐1β‐induced inflammatory responses in chondrocytes from osteoarthritis patients: Involvement of HIF‐1α pathway

It has been reported that vitexin has anti‐inflammatory effects in osteoarthritis (OA) rats. However, the effects of vitexin on interleukins‐1β (IL‐1β)‐stimulated OA patient‐derived chondrocytes have not been reported. The purpose of this study was to investigate the anti‐inflammatory effects of vitexin on IL‐1β‐stimulated human osteoarthritis chondrocytes and to reveal the involvement of hypoxia‐inducible factor 1α (HIF‐1α) pathway. Enzyme‐linked immunosorbent assay, quantitative real‐time PCR and Western blotting assays were employed. ELISA results demonstrated that the proinflammatory cytokine levels of interleukins‐6 (IL‐6) and tumour necrosis factor α (TNF‐α) in the serum and synovial fluid and HIF‐1α level in the synovial fluid were significantly elevated in OA patients compared to normal healthy subjects. Moreover, the Western blotting results indicated that the protein expression of HIF‐1α was significantly higher in the cartilage tissues of OA patients. OA patient‐derived chondrocytes were stimulated by IL‐1β and treated with different concentration of vitexin for 24 hours. Vitexin showed no cytotoxicity and increased the survival of chondrocytes under IL‐1β stimulation. Vitexin suppressed IL‐1β‐induced production of NO and prostaglandin E2 (PGE₂) in chondrocytes culture. The treatment of vitexin significantly inhibited IL‐1β‐induced expressions of proinflammatory cytokine levels of IL‐6, TNF‐α, matrix metalloproteinase (MMP)‐1, MMP‐3 and MMP‐13. Furthermore, Western blotting results demonstrated that HIF‐1α is involved in vitexin's protective effects on IL‐1β‐stimulated injuries in OA patient‐derived chondrocytes. Our study demonstrates that vitexin alleviates IL‐1β‐induced inflammatory responses in chondrocytes from osteoarthritis patients, which may be attributed partly to the inhibition of HIF‐1α pathway.     

Hypoxic inactivation of glycogen synthase kinase‐3β promotes gastric tumor growth and angiogenesis by facilitating hypoxia‐inducible factor‐1 signaling

Since the molecular mechanism of hypoxic adaptation in cancer cells is cell‐type specific, we investigated whether glycogen synthase kinase‐3β (GSK‐3β) activation is involved in hypoxia‐induced gastric tumor promotion. Stable gastric cancer cell lines (SNU‐638, SNU‐484, MKN1, and MKN45) were cultured under hypoxic conditions. Cells overexpressing wild‐type GSK‐3β (WT‐GSK‐3β) or kinase‐dead mutant of GSK‐3β (KD‐GSK‐3β) were generated and used for cell culture and animal studies. In cell culture experiments, hypoxia decreased GSK‐3β activation in gastric cancer cells. Cell viability and the expressions of HIF‐1α protein and VEGF mRNA in gastric cancer cells were higher in KD‐GSK‐3β transfectants than in WT‐GSK‐3β transfectants under hypoxic conditions, but not under normoxic conditions. Gastric cancer xenografts showed that tumor growth, microvessel area, HIF‐1α activation, and VEGF expression were higher in KD‐GSK‐3β tumors than in WT‐GSK‐3β tumors in vivo. In addition, the expression of hypoxia‐induced HIF‐1α protein was regulated by GSK‐3β at the translational level. Our data suggest that GSK‐3β is involved in hypoxic adaptation of gastric cancer cells as an inhibitory upstream regulator of the HIF‐1α/VEGF signaling pathway.     

Hypoxia‐inducible factor‐1α perpetuates synovial fibroblast interactions with T cells and B cells in rheumatoid arthritis

Synovial fibroblast hyperplasia, T‐cell hyperactivity, B‐cell overactivation, and the self‐perpetuating interactions among these cell types are major characteristics of rheumatoid arthritis (RA). The inflamed joints of RA patients are hypoxic, with upregulated expression of hypoxia‐inducible factor‐1α (HIF‐1α) in RA synovial fibroblasts (RASFs). It remains unknown whether HIF‐1α regulates interactions between RASFs and T cells and B cells. We report here that HIF‐1α promotes the expression of inflammatory cytokines IL‐6, IL‐8, TNF‐α, and IL‐1β, and cell–cell contact mediators IL‐15, vascular cell adhesion molecule (VCAM)‐1, thrombospondin (TSP)‐1, and stromal cell‐derived factor (SDF)‐1 in RASFs. Furthermore, HIF‐1α perpetuates RASF‐mediated inflammatory Th1‐ and Th17‐cell expansion while differentially inhibiting regulatory B10 and innate‐like B cells, leading to increased IFN‐γ, IL‐17, and IgG production and decreased protective natural IgM secretion. Our findings suggest that HIF‐1α perpetuates the interactions between RASFs and T cells and B cells to induce inflammatory cytokine and autoantibody production, thus exacerbating the severity of RA. Targeting HIF‐1α may provide new therapeutic strategies for overcoming this persistent disease.     

Apparent diffusion coefficients in prostate cancer: correlation with molecular markers Ki‐67, HIF‐1α and VEGF

Prostate cancer (PCa) is the second most common cancer in men. The Gleason score (GS) and biomarkers play important roles in the diagnosis and treatment of patients with PCa. The purpose of this study was to investigate the relationship between the apparent diffusion coefficient (ADC) and the molecular markers Ki‐67, hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) in PCa. Thirty‐nine patients with 39 lesions, who had been diagnosed with PCa, were enrolled in this study. All patients underwent diffusion‐weighted magnetic resonance imaging (DW‐MRI) (b = 800 s/mm²). The expression of Ki‐67, HIF‐1α and VEGF was assessed by immunohistochemistry. Statistical analysis was applied to analyze the association between ADC and prostate‐specific antigen (PSA), GS and the expression of Ki‐67, HIF‐1α and VEGF. The group differences in ADC among different grades of Ki‐67, HIF‐1α and VEGF were also analyzed. The mean ± standard deviation of ADC was (0.76 ± 0.27) × 10^?3 mm²/s. ADC correlated negatively with PSA and GS (p < 0.05). The Ki‐67 staining index (SI), HIF‐1α expression and VEGF expression in PCa were correlated inversely with ADC, controlling for age (r = –0.332, p < 0.05; r = ?0.662, p < 0.0005; and r = ?0.714, p < 0.0005, respectively). ADC showed a significant difference among different grades of Ki‐67 (F = 9.164, p = 0.005), HIF‐1α (F = 40.333, p < 0.0005) and VEGF (F = 22.048, p < 0.0005). In conclusion, ADC was correlated with PSA, GS, and Ki‐67, HIF‐1α and VEGF expression in patients with PCa. ADC may be used to evaluate tumor proliferation, hypoxia and angiogenesis in PCa.     

Hypoxia inducible factor‐1α‐induced interleukin‐33 expression in intestinal epithelia contributes to mucosal homeostasis in inflammatory bowel disease

Intestinal epithelial cells (IECs), an important barrier to gut microbiota, are subject to low oxygen tension, particularly during intestinal inflammation. Hypoxia inducible factor‐1α (HIF‐1α) is expressed highly in the inflamed mucosa of inflammatory bowel disease (IBD) and functions as a key regulator in maintenance of intestinal homeostasis. However, how IEC‐derived HIF‐1α regulates intestinal immune responses in IBD is still not understood completely. We report here that the expression of HIF‐1α and IL‐33 was increased significantly in the inflamed mucosa of IBD patients as well as mice with colitis induced by dextran sulphate sodium (DSS). The levels of interleukin (IL)?33 were correlated positively with that of HIF‐1α. A HIF‐1α‐interacting element was identified in the promoter region of IL‐33, indicating that HIF‐1α activity regulates IL‐33 expression. Furthermore, tumour necrosis factor (TNF) facilitated the HIF‐1α‐dependent IL‐33 expression in IEC. Our data thus demonstrate that HIF‐1α‐dependent IL‐33 in IEC functions as a regulatory cytokine in inflamed mucosa of IBD, thereby regulating the intestinal inflammation and maintaining mucosal homeostasis.     

HIF‐1α triggers long‐lasting glutamate excitotoxicity via system xc− in cerebral ischaemia–reperfusion

Hypoxia‐inducible factor 1α (HIF ‐1α) controls many genes involved in physiological and pathological processes. However, its roles in glutamatergic transmission and excitotoxicity are unclear. Here, we proposed that HIF ‐1α might contribute to glutamate‐mediated excitotoxicity during cerebral ischaemia–reperfusion (CIR ) and investigated its molecular mechanism. We showed that an HIF ‐1α conditional knockout mouse displayed an inhibition in CIR ‐induced elevation of extracellular glutamate and N ‐methyl‐d ‐aspartate receptor (NMDAR ) activation. By gene screening for glutamate transporters in cortical cells, we found that HIF ‐1α mainly regulates the cystine–glutamate transporter (system x_c^?) subunit xCT by directly binding to its promoter; xCT and its function are up‐regulated in the ischaemic brains of rodents and humans, and the effects lasted for several days. Genetic deletion of xCT in cortical cells of mice inhibits either oxygen glucose deprivation/reoxygenation (OGDR ) or CIR ‐mediated glutamate excitotoxicity in vitro and in vivo . Pharmaceutical inhibition of system x_c^? by a clinically approved anti‐cancer drug, sorafenib, improves infarct volume and functional outcome in rodents with CIR and its therapeutic window is at least 3 days. Taken together, these findings reveal that HIF ‐1α plays a role in CIR ‐induced glutamate excitotoxicity via the long‐lasting activation of system x_c^?‐dependent glutamate outflow and suggest that system x_c^? is a promising therapeutic target with an extended therapeutic window in stroke. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Different cytokine production profiles of γδ T cell clones: Relation to inflammatory arthritis

This study was performed to investigate whether γδ T cells could also be divided into subsets, identified by a cytokine profile, as described for αβ T helper (Th) cell subsets. Cytokine production was studied in 22 γδ T cell clones obtained from the synovial fluid and peripheral blood of one patient with inflammatory arthritis and compared to that of 26 αβ T cell clones of the same and different patients. Interferon-γ (IFN-γ) was produced by 18 (82%) and interleukin-4 (IL-4) by 17 (77%) out of 22 γδ T cell clones, respectively. In contrast, IL-10 was not produced, except at very low level in one case. The mean levels of IL-4 were lower for clones derived from synovial fluid. When considering the production of IFN-γ as an indicator of Th1 and that of IL-4 as an indicator of Th2, respectively, the most common pattern was a γδTh1-like pattern, with the combination of high levels of IFN-γ and low levels of IL-4. This pattern was found in Vδ1⁺ clones, all from synovial fluid. Additional patterns were also observed: a mixed, probably γδ Th0-like pattern with a more balanced production of both IFN-γ and IL-4; a γδTh1 pattern with the production of IFN-γ alone; a γδTh2 pattern with the production of IL-4 alone. These three patterns were also seen in blood γδ T cells which were all Vδ2, indicating that these patterns were independent of the γδ phenotype. γδ T cell clones produced lower levels of IFN-γ (p = 0.001) and higher levels of IL-4 than αβ clones (p < 0.02). These differences in cytokine production between αβ and γδ subsets and within these subsets may contribute to their respective role in chronic inflammation.     

Molecular characterization and clinical presentation of HKαα and anti‐HKαα alleles in southern Chinese subjects

The HKαα allele is a rearrangement occurring in the α‐globin gene cluster containing both the ‐α^3.7 and ααα^anti4.2 unequal crossover junctions. The anti‐HKαα allele is the reciprocal product containing both the ‐α^4.2 and ααα^anti3.7 unequal crossover junctions, which had been predicted but had not been detected previously. The phenotypic feature and population frequency of these two unusual alleles were not described. We report the identification of nine individuals carrying the HKαα allele and two individuals carrying the anti‐HKαα allele in southern China and describe their phenotype and haplotype data. The molecular structures of HKαα allele and anti‐HKαα allele were confirmed by two‐round nested polymerase chain reaction assay. The mechanism of origin of both alleles is related to probably simultaneous double crossover. Heterozygotes of HKαα or anti‐HKαα allele show a normal hematological phenotype. Finally, we report the carrier rates of these both alleles in the Guangxi Zhuang Autonomous Region of southern China, namely, ∼0.07% for the HKαα allele and ∼0.02% for the anti‐HKαα allele.     

22‐S‐Hydroxycholesterol protects against ethanol‐induced liver injury by blocking the auto/paracrine activation of MCP‐1 mediated by LXRα

Chronic ethanol consumption causes hepatic steatosis and inflammation, which are associated with liver hypoxia. Monocyte chemoattractant protein‐1 (MCP‐1) is a hypoxia response factor that determines recruitment and activation of monocytes to the site of tissue injury. The level of MCP‐1 is elevated in the serum and liver of patients with alcoholic liver disease (ALD); however, the molecular details regarding the regulation of MCP‐1 expression are not yet understood completely. Here, we show the role of liver X receptor α (LXRα) in the regulation of MCP‐1 expression during the development of ethanol‐induced fatty liver injury, using an antagonist, 22‐S‐hydroxycholesterol (22‐S‐HC). First, administration of 22‐S‐HC attenuated the signs of liver injury with decreased levels of MCP‐1 and its receptor CCR2 in ethanol‐fed mice. Second, hypoxic conditions or treatment with the LXRα agonist GW3965 significantly induced the expression of MCP‐1, which was completely blocked by treatment with 22‐S‐HC or infection by shLXRα lentivirus in the primary hepatocytes. Third, over‐expression of LXRα or GW3965 treatment increased MCP‐1 promoter activity by increasing the binding of hypoxia‐inducible factor‐1α to the hypoxia response elements, together with LXRα. Finally, treatment with recombinant MCP‐1 increased the level of expression of LXRα and LXRα‐dependent lipid droplet accumulation in both hepatocytes and Kupffer cells. These data show that LXRα and its ligand‐induced up‐regulation of MCP‐1 and MCP‐1‐induced LXRα‐dependent lipogenesis play a key role in the autocrine and paracrine activation of MCP‐1 in the pathogenesis of alcoholic fatty liver disease, and that this activation may provide a promising new target for ALD therapy.Copyright © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

The stimulatory effect of α‐melanotropin on progesterone release from rat granulosa cells is inhibited by interleukin‐1β and by tumour necrosis factor‐α

Aim: Several studies have shown that a variety of peptides and cytokines are involved in ovarian regulatory mechanisms; however, their exact function is still unclear. In this work we study whether the administration of peptide α‐melanotropin and the cytokines interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) on their own modify the release of progesterone in cultured granulosa cells (GC) from pro‐oestrous rats. We also investigate an interaction between these cytokines and α‐melanotropin in the modulation of progesterone secretion. Methods: Granulosa cells were collected from the ovaries of female Wistar rats and cultured for up to 24 h in the presence of different concentrations of α‐melanotropin, cytokines or a combination of both. Progesterone concentration was measured by radioimmunoassay. Results: The addition of α‐melanotropin in a dose of 0.01 and 0.1 mm had no effect on progesterone release, whereas a dose of 1 mm significantly increased progesterone release (P < 0.01) compared with the control culture. Progesterone release was not modified when different concentrations of interleukin‐1β or TNF‐α were added to the cell cultures. However, when interleukin‐1β or TNF‐α were added simultaneously with 1 μm α‐melanotropin, a significant reduction (P < 0.01 for interleukin‐1β and P < 0.05 for TNF‐α) of the steroid release was found with respect to the α‐melanotropin‐treated group. Conclusions: These results lead us to suggest that, although α‐melanotropin stimulates progesterone release in pre‐ovulatory GC, this effect is blocked by the presence of interleukin‐1β or TNF‐α.     

Interleukin‐27 suppresses osteoclastogenesis via induction of interferon‐γ

Interleukin (IL)‐27 is a heterodimeric cytokine that is known to have both stimulatory and inhibitory functions during immune responses. We investigated the effects of IL‐27 on arthritis and bone erosion in the murine collagen‐induced arthritis (CIA) model. We demonstrate that the inhibitory effect of IL‐27 on osteoclastogenesis is associated with interferon‐γ (IFN‐γ) production by using an IFN‐γ knockout mouse model. The IL‐27‐Fc was injected into both CIA and IFN‐γ‐deficient mice. The effects of IL‐27‐Fc on osteoclast differentiation were evaluated both in vitro and in vivo. The IL‐27‐Fc‐injected mice showed significantly lower arthritis indices and fewer tartrate‐resistant acid‐phosphatase‐positive osteoclasts in their joint tissues than untreated mice. Interleukin‐27 inhibited osteoclastogenesis from bone marrow‐derived mononuclear cells in vitro, which was counteracted by the addition of anti‐IFN‐γ antibody. The IL‐27‐Fc did not affect arthritis in IFN‐γ knockout mice. Interleukin‐27 also suppressed osteoclast differentiation in human and intriguingly, it could promote the expression of IFN‐γ on priming osteoclasts. These results imply that IL‐27 suppressed the generation of CIA and osteoclastogenesis, which were mediated by the induction of IFN‐γ.