Inhibition of Hypoxia Inducible Factor 1α by siRNA‐Induced Apoptosis in Human Retinoblastoma Cells

Hypoxia, which activates the hypoxia inducible factor 1α (HIF‐1α), is an essential feature of retinoblastoma (RB) and contributes to poor prognosis and resistance to conventional therapy. In this study, the effect of HIF‐1α knockdown by small interfering RNA (siRNA) on cell proliferation, apoptosis, and apoptotic pathways of human Y‐79 RB cells was first investigated. Exposure to hypoxia induced the increased expression of HIF‐1α both in mRNA and protein levels. Then, knockdown of HIF‐1α by siRNA_HIF‐1α resulted in inhibition of cell proliferation and induced cell apoptosis in human Y‐79 RB cells under both normoxic and hypoxic conditions, with hypoxic conditions being more sensitive. Furthermore, knockdown of HIF‐1α could enhance hypoxia‐induced slight increase of Bax/Bcl‐2 ratio and activate caspase‐9 and caspase‐3. These results together indicated that suppression of HIF‐1α expression may be a promising strategy for the treatment of human RB in the future.     

Glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced Müller glial galectin‐1 expression via HIF‐1α destabilization

Galectin‐1/LGALS1, a newly recognized angiogenic factor, contributes to the pathogenesis of diabetic retinopathy (DR). Recently, we demonstrated that glucocorticoids suppressed an interleukin‐1β‐driven inflammatory pathway for galectin‐1 expression in vitro and in vivo. Here, we show glucocorticoid‐mediated inhibitory mechanism against hypoxia‐inducible factor (HIF)‐1α‐involved galectin‐1 expression in human Müller glial cells and the retina of diabetic mice. Hypoxia‐induced increases in galectin‐1/LGALS1 expression and promoter activity were attenuated by dexamethasone and triamcinolone acetonide in vitro. Glucocorticoid application to hypoxia‐stimulated cells decreased HIF‐1α protein, but not mRNA, together with its DNA‐binding activity, while transactivating TSC22 domain family member (TSC22D)3 mRNA and protein expression. Co‐immunoprecipitation revealed that glucocorticoid‐transactivated TSC22D3 interacted with HIF‐1α, leading to degradation of hypoxia‐stabilized HIF‐1α via the ubiquitin‐proteasome pathway. Silencing TSC22D3 reversed glucocorticoid‐mediated ubiquitination of HIF‐1α and subsequent down‐regulation of HIF‐1α and galectin‐1/LGALS1 levels. Glucocorticoid treatment to mice significantly alleviated diabetes‐induced retinal HIF‐1α and galectin‐1/Lgals1 levels, while increasing TSC22D3 expression. Fibrovascular tissues from patients with proliferative DR demonstrated co‐localization of galectin‐1 and HIF‐1α in glial cells partially positive for TSC22D3. These results indicate that glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced retinal glial galectin‐1/LGALS1 expression via HIF‐1α destabilization, highlighting therapeutic implications for DR in the era of anti‐vascular endothelial growth factor treatment.     

Ferulic acid improves self‐renewal and differentiation of human tendon‐derived stem cells by upregulating early growth response 1 through hypoxia

We aimed to investigate the potential beneficial effect of ferulic acid (FA) on stemness of human tendon‐derived stem cells (hTSCs) in vitro and to elucidate the underlying molecular mechanism. The self‐renewal ability of hTSCs was evaluated by colony formation and cell proliferation was determined by CCK‐8 kit. Adipogenesis, osteogenesis, and chondrogenesis were determined by Oil Red O, Alizarin Red, and Alcian Blue stainings, respectively. Relative mRNA levels of PPARγ, Col2A1, Acan, Runx2, HIF1α, and EGR1 were measured with real‐time PCR. Protein levels of HIF1α and EGR1 were detected by western blot. Direct binding of HIF1α with EGR1 promoter was analyzed by ChIP assay. Hypoxia‐induced expression of EGR1 was interrogated by luciferase reporter assay. We demonstrated that FA treatment improved both self‐renewal ability and multi‐differentiation potential of hTSCs. FA induced hypoxia which in turn upregulated EGR1 expression via direct association with its hypoxia response element consensus sequence. Furthermore, we showed that both HIF1α and EGR1 were required for the enhancing effects of FA on hTSC self‐renewal and differentiation. We hereby characterize the beneficial effect of FA on the stemness of hTSCs and highlight the critical role of HIF1α‐EGR1 axis in this process.     

Effect of arachidonic acid on hypoxia‐induced IL‐6 production in mouse ES cells: Involvement of MAPKs,NF‐κB,and HIF‐1α

This study examined the role of arachidonic acid (AA) in hypoxia‐induced production of interleukin (IL)‐6 and its related signaling pathways in mouse embryonic stem (ES) cells. Hypoxia with AA induced IL‐6 production, which was mediated by reactive oxygen species (ROS). In addition, hypoxia increased the levels of p38 mitogen‐activated protein kinases (MAPKs) and stress‐activated protein kinase/c‐jun NH₂‐terminal kinase (SAPK/JNK) phosphorylation, which were blocked by antioxidant (vitamin C). Inhibition of p38 MAPK and SAPK/JNK blocked hypoxia‐ or hypoxia with AA‐induced nuclear factor‐kappa B (NF‐κB) activation. Furthermore, hypoxia‐induced increase in hypoxia‐inducible factor‐1α (HIF‐1α) expression was regulated by NF‐κB activation. Consequently, the increased HIF‐1α expression induced activation of matrix metalloproteinase (MMP)‐2 and MMP‐9. The expression of each signaling molecule stimulated an increase in IL‐6 production that was greater in hypoxic conditions with AA than with hypoxia alone. Finally, inhibition of IL‐6 production using IL‐6 antibody or soluble IL‐6 receptor attenuated the hypoxia‐induced increases in DNA synthesis of mouse ES cells. In conclusion, AA potentiates hypoxia‐induced IL‐6 production through the MAPKs, NF‐κB, and HIF‐1α pathways in mouse ES cells. J. Cell. Physiol. 222: 574–585, 2010. © 2009 Wiley‐Liss, Inc.     

Mutual regulation between butyrate and hypoxia‐inducible factor‐1α in epithelial cell promotes expression of tight junction proteins

Inflammatory bowel disease is a kind of multi‐aetiological chronic disease that is driven by multidimensional factors. Hypoxia‐inducible factor‐1α (HIF‐1α) plays an important role in anti‐inflammatory and cellular responses to hypoxia. Previous studies have found that B or T‐cell‐specific HIF‐1α knock out mice exhibit severe colonic inflammation. However, we know very little about other functions of HIF‐1α in intestinal epithelial cells (IECs). In our study, HIF‐1α^ΔIEC mice were used to study the function of HIF‐1α in IECs. HIF‐1α was knocked down in Caco‐2 cells by transfection with a small interfering (si) RNA. Immunohistochemical staining and western blotting were used to detect the expression of zonula occluden‐1 (ZO‐1) and Occludin. The content of colon was harvested for high‐performance liquid chromatography analysis to examine the levels of butyrate in the gut. Our research found that HIF‐1α played a protective role in dextran sulphate sodium‐induced colitis, which was partly due to its regulation of tight junction (TJ) protein expression. Further study revealed that HIF‐1α mediated TJ proteins levels by moderating the content of butyrate. Moreover, we found that butyrate regulated TJ protein expression, which is dependent on HIF‐1α. These results indicated that there is a mutual regulatory mechanism between butyrate and HIF‐1α, which has an important role in the maintenance of barrier function of the gastrointestinal tract.     

Up-regulation of glyceraldehyde-3-phosphate dehydrogenase gene expression by HIF-1 activity depending on Sp1 in hypoxic breast cancer cells

Hypoxia up-regulates the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a cell type-specific manner. It is unknown whether this occurs in breast cancer. Here, we report that hypoxia up-regulates the GAPDH gene expression through breast cancer-specific molecular mechanisms in MCF-7 cells. Mutation analysis identified a novel hypoxia response element (HRE), in addition to the HRE found previously in prostate cancer LNCaP cells. Knockdown and overexpression of hypoxia-inducible factor (HIF)-1α indicated that HIF-1 contributed to the up-regulation of GAPDH gene expression by hypoxia. Although chromatin immunoprecipitation (ChIP) and plasmid immunoprecipitation analyses revealed the presence of HIF-1α on the novel HRE in both hypoxic cell lines, a mutation in either the novel HRE or its 3′-flanking GC-box resulted in a reduction of hypoxia-increased GAPDH promoter activity only in MCF-7 cells. ChIP analysis showed that Sp1 bound to the GC-box in MCF-7 cells, but not in LNCaP cells, in normoxia and hypoxia. Knockdown of Sp1 reduced hypoxia-increased promoter activity and expression level of GAPDH in MCF-7 cells. These results indicate that in MCF-7 cells, the activation of HIF-1 on the novel HRE contributes to the breast cancer-specific hypoxic induction of GAPDH gene expression and absolutely depends on the presence of Sp1 on the GC-box.     

Effect of HIF1α on Foxp3 expression in CD4+CD25− T lymphocytes

The aim of the present study was to investigate the effect of HIF1α on Foxp3 expression in CD4⁺CD25^? T lymphocytes. CD4⁺CD25^? T lymphocytes were sorted from PBMC using a CD4⁺CD25⁺ regulatory T cell isolation kit. Lentivirus containing lentiviral vector that overexpressed HIF1α (HIF‐lenti) and those containing empty expression vector (control‐lenti) were produced. Meanwhile, lentivirus that contained lentiviral vector that suppressed HIF1α expression (siHIF‐lenti) and those containing control vector (sicontrol‐lenti) were also generated. The sorted CD4⁺CD25^? T lymphocytes were infected with HIF‐lenti, control‐lenti, siHIF‐lenti, and sicontrol‐lenti, respectively. Approximately 72 hr after transduction, real‐time PCR and Western blot were carried out to analyze the RNA and protein expression level of HIF1α and Foxp3. CD4⁺CD25^? T lymphocytes cultured under 21% O₂, 5% CO₂ (normoxia) and 1% O₂, 5% CO₂ (hypoxia) were used as control. Our results showed that overexpression of HIF1α increased both mRNA and protein expression of Foxp3 and, meanwhile, suppression of HIF1α expression by RNAi could reverse high Foxp3 expression in CD4⁺CD25^? T lymphocytes caused by hypoxic culture. These results suggested that hypoxia could stimulate Foxp3 expression by increasing HIF1α expression in CD4⁺ T lymphocytes which may promote CD4⁺ T lymphocytes to convert to Treg.

     

Qiliqiangxin attenuates hypoxia‐induced injury in primary rat cardiac microvascular endothelial cells via promoting HIF‐1α‐dependent glycolysis

Protection of cardiac microvascular endothelial cells (CMECs) against hypoxia injury is an important therapeutic strategy for treating ischaemic cardiovascular disease. In this study, we investigated the effects of qiliqiangxin (QL) on primary rat CMECs exposed to hypoxia and the underlying mechanisms. Rat CMECs were successfully isolated and passaged to the second generation. CMECs that were pre‐treated with QL (0.5 mg/mL) and/or HIF‐1α siRNA were cultured in a three‐gas hypoxic incubator chamber (5% CO₂, 1% O₂, 94% N₂) for 12 hours. Firstly, we demonstrated that compared with hypoxia group, QL effectively promoted the proliferation while attenuated the apoptosis, improved mitochondrial function and reduced ROS generation in hypoxic CMECs in a HIF‐1α‐dependent manner. Meanwhile, QL also promoted angiogenesis of CMECs via HIF‐1α/VEGF signalling pathway. Moreover, QL improved glucose utilization and metabolism and increased ATP production by up‐regulating HIF‐1α and a series of glycolysis‐relevant enzymes, including glucose transport 1 (GLUT1), hexokinase 2 (HK2), 6‐phosphofructokinase 1 (PFK1), pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). Our findings indicate that QL can protect CMECs against hypoxia injury via promoting glycolysis in a HIF‐1α‐dependent manner. Lastly, the results suggested that QL‐dependent enhancement of HIF‐1α protein expression in hypoxic CMECs was associated with the regulation of AMPK/mTOR/HIF‐1α pathway, and we speculated that QL also improved HIF‐1α stabilization through down‐regulating prolyl hydroxylases 3 (PHD3) expression.