BACKGROUND

Recurrent airway obstruction (RAO, also known as equine heaves) is an inflammatory condition caused by exposure of susceptible horses to organic dusts in hay. The immunological processes responsible for the development and the persistence of airway inflammation are still largely unknown. Hypoxia-inducible factor (Hif) is mainly known as a major regulator of energy homeostasis and cellular adaptation to hypoxia. More recently however, Hif also emerged as an essential regulator of innate immune responses. Here, we aimed at investigating the potential involvement of Hif1-α in myeloid cells in horse with recurrent airway obstruction.

RESULTS

In vitro, we observed that Hif is expressed in equine myeloid cells after hay dust stimulation and regulates genes such as tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8) and vascular endothelial growth factor A (VEGF-A). We further showed in vivo that airway challenge with hay dust upregulated Hif1-α mRNA expression in myeloid cells from the bronchoalveolar lavage fluid (BALF) of healthy and RAO-affected horses, with a more pronounced effect in cells from RAO-affected horses. Finally, Hif1-α mRNA expression in BALF cells from challenged horses correlated positively with lung dysfunction.

CONCLUSIONS

Taken together, our results suggest an important role for Hif1-α in myeloid cells during hay dust-induced inflammation in horses with RAO. We therefore propose that future research aiming at functional inactivation of Hif1 in lung myeloid cells could open new therapeutic perspectives for RAO.

How the immune system is negatively affected by sepsis is not fully understood. In this issue of Immunity, Shalova et al. (2015) show that during human sepsis monocytes upregulate hypoxia-inducible factor-α (HIF1-α) activity and acquire an immunosuppressive phenotype while retaining anti-bacterial and wound-healing properties.

Hypoxia is one of the basic characteristics of the colorectal cancer and HIF1 plays a central role in tumor hypoxia adaptation and controls the expression of a variety of genes. But it had not reached a comprehensive understanding of them in colorectal cancer in hypoxia. This study silenced HIF1-alpha, which is the regulatory subunit of HIF1, and established two dimensional gel electrophoresis profiles for human SW480siHIF1-alpha and SW480N cells to identified proteins with differential expressions. Ten down-regulated proteins and nine up-regulated proteins were identified by HPLC-Chip-MS/MS. Down-regulated proteins including PLD2, ANXA2. HIF1-alpha had a clear positive correlation with PLD2 and ANXA2 in colon adenocarcinoma samples. There proteins might have a direct or indirect contact HIF1-alpha, and play a critical role in apoptosis, signal transduction and hypoxia adaptation.

OBJECTIVE

The transcription factor MITF (microphthalmia-associated transcription factor) is known to induce expression of hypoxia-inducible factor (HIF1-α), which is involved in renal carcinogenesis. The MITF p.E318K mutation leads to deficient SUMOylation of MITF, resulting in enhanced activation of its target genes. A case-control study on melanoma patients who coincidentally were affected by renal cell carcinoma (RCC) has revealed an elevated risk for mutation carriers to be affected by one or both of these malignancies, suggesting a possible role for MITF p.E318K in renal carcinogenesis. The same study described an MITF mutation frequency of 1.5% in a small cohort of sporadic RCC, but comprehensive data on sporadic renal cell tumors are missing. We therefore tested a large cohort of sporadic renal tumors for MITF p.E318K mutation status.

METHODS

Genomic DNA was extracted from 426 formalin-fixed, paraffin-embedded sporadic renal tumors that had been graded according to the 2004 WHO classification of renal tumors and staged according to the 2002 TNM classification. The tumor cohort was enriched with papillary and chromophobe RCC, and also contained benign oncocytomas. DNA was tested for MITF p.E318K by pyrosequencing.

RESULTS

Of 403 analyzable tumors, 402 renal tumors were wild-type ones, and only 1 case showed the MITF p.E318K mutation. This tumor was a clear-cell RCC (pT3b N0 M0 G3 according to the TNM classification 2002). The affected patient was male, 61 years old, and had no known coexisting malignancies.

CONCLUSIONS

The MITF p.E318K mutation does not appear to play a major role in sporadic RCC carcinogenesis, but is possibly restricted to a rare subpopulation of inherited RCC.

Yeast Hif1 [Hat1 (histone acetyltransferase 1)-interacting factor], a homologue of human NASP (nuclear autoantigenic sperm protein), is a histone chaperone that is involved in various protein complexes which modify histones during telomeric silencing and chromatin reassembly. For elucidating the structural basis of Hif1, in the present paper we demonstrate the crystal structure of Hif1 consisting of a superhelixed TPR (tetratricopeptide repeat) domain and an extended acid loop covering the rear of TPR domain, which represent typical characteristics of SHNi-TPR [Sim3 (start independent of mitosis 3)-Hif1-NASP interrupted TPR] proteins. Our binding assay indicates that Hif1 could bind to the histone octamer via histones H3 and H4. The acid loop is shown to be crucial for the binding of histones and may also change the conformation of the TPR groove. By binding to the core histone complex Hif1 may recruit functional protein complexes to modify histones during chromatin reassembly.

Hypoxia-inducible factor (HIF-1α) protein is over-expressed in many human cancers and is a major cause of resistance to drugs. HIF-1α up-regulation decreases the effectiveness of several anticancer agents, including 5-fluorouracil (5-FU), because it induces the expression of drug efflux transporters, alters DNA repair mechanisms and modifies the balance between pro- and antiapoptotic factors. These findings suggest that inhibition of HIF-1α activity may sensitize cancer cells to cytotoxic drugs. We previously reported that l-carnosine reduces HIF-1α expression by inhibiting the proliferation of colon cancer cells. In the present study we investigated the effect of l-carnosine on HT29 colon cancer cells with acquired resistance to 5-FU. We found that l-carnosine reduces colon cancer cell viability, decreases HIF-1α and multi-drug resistant protein MDR1-pg expression, and induces apoptosis. Moreover, the l-carnosine/5-FU combination lowers the expression of some chemoresistance markers. The combination index evaluated in vitro on the HT29-5FU cell line by median drug effect analysis reveals a significant synergistic effect.

BACKGROUND

Neuroendocrine (NE) differentiation in prostate cancer has been correlated with a poor prognosis and hormone refractory disease. In a previous report, we demonstrated the presence of immunoreactive cytoplasmic hypoxia inducible factor 1alpha (HIF1alpha), in both benign and malignant NE prostate cells. HIF1alpha and HIF1beta are two subunits of HIF1, a transcription factor important for angiogenesis. The aim of this study was to elucidate whether the cytoplasmic stabilization of HIF1alpha in androgen independent NE differentiated prostate cancer is due to the presence of certain HIF1alpha isoforms.

METHODS

We studied the HIF1alpha isoforms present in 8 cases of benign prostate hyperplasia (BPH) and 43 cases of prostate cancer with and without NE differentiation using RT-PCR, sequencing analysis, immunohistochemistry and in situ hybridization.

RESULTS

We identified multiple isoforms in both benign and malignant prostate tissues. One of these isoforms, HIF1alphaas previously reported to be testis specific, was found in 86% of NE-differentiated prostate tumors, 92% of HIF1alpha immunoreactive prostate tumors and 100% of cases of benign prostate hyperplasia. Immunohistochemistry and in situ hybridization results showed that this isoform corresponds to the cytoplasmic HIF1alpha present in androgen-independent NE cells of benign and malignant prostate tissue and co-localizes with immunoreactive cytoplasmic HIF1beta.

CONCLUSIONS

Our results indicate that the cytoplasmic stabilization of HIF1alpha in NE-differentiated cells in benign and malignant prostate tissue is due to presence of an HIF1alpha isoform, HIF1alphaalization of this isoform with HIF1beta indicates that the HIF1alphaHIF1beta in the cytoplasm.

Hypoxia-inducible factor 1α (HIF-1α) is a master regulator of oxygen homeostasis. Under hypoxia, the active HIF1-α subunits are mainly regulated through increased protein stabilization. Little is known concerning HIF-1α transcriptional regulation. Nuclear respiratory factor 1 (NRF-1) is a DNA-binding transcription factor that regulates mitochondrial biogenesis. In this study, we showed that NRF-1was a repressor of HIF-1α. The cellular depletion of NRF-1 by siRNA targeting leads to increased HIF-1αtranscriptional activity. EMSA, ChIP and luciferase activity allowed the identification of two functional NRF-1 binding sites within HIF-1α promoter. This study therefore identifies NRF-1 as a novel regulator of HIF-1α. © 2016 IUBMB Life, 68(9):748-755, 2016.

Neoangiogenesis is a fundamental process which helps to meet energy requirements, tissue growth, and wound healing. Although previous studies showed that Peroxisome proliferator-activated receptor (PPAR-γ) regulates neoangiogenesis via upregulation of vascular endothelial growth factor (VEGF), and both VEGF and PPAR-γ expressions were inhibited during hyperhomocysteinemic (HHcy), whether these two processes could trigger pathological effects in skeletal muscle via compromising neoangiogenesis has not been studied yet. Unfortunately, there are no treatment options available to date for ameliorating HHcy-mediated neoangiogenic defects. Hydrogen sulfide (H2 S) is a novel gasotransmitter that can induce PPAR-γ levels. However, patients with cystathionine-β-synthase (CBS) mutation(s) cannot produce a sufficient amount of H2 S. We hypothesized that exogenous supplementation of H2 S might improve HHcy-mediated poor neoangiogenesis via the PPAR-γ/VEGF axis. To examine this, we created a hind limb femoral artery ligation (FAL) in CBS+/- mouse model and treated them with GYY4137 (a long-acting H2 S donor compound) for 21 days. To evaluate neoangiogenesis, we used barium sulfate angiography and laser Doppler blood flow measurements in the ischemic hind limbs of experimental mice post-FAL to assess blood flow. Proteins and mRNAs levels were studied by Western blots and qPCR analyses. HIF1-α, VEGF, PPAR-γ and p-eNOS expressions were attenuated in skeletal muscle of CBS+/- mice after 21 days of FAL in comparison to wild-type (WT) mice, that were improved via GYY4137 treatment. We also found that the collateral vessel density and blood flow were significantly reduced in post-FAL CBS+/- mice compared to WT mice and these effects were ameliorated by GYY4137. Moreover, we found that plasma nitrite levels were decreased in post-FAL CBS+/- mice compared to WT mice, which were mitigated by GYY4137 supplementation. These results suggest that HHcy can inhibit neoangiogenesis via antagonizing the angiogenic signal pathways encompassing PPAR-γ/VEGF axis and that GYY4137 could serve as a potential therapeutic to alleviate the harmful metabolic effects of HHcy conditions.

We reported previously that supplementation with apple pectin, a dietary fiber, reduced myocardial injury in a rat model of ischemia-reperfusion. In this study, we further investigated an arabinogalactan, one of the constituent polysaccharides of pectin, to determine which domains comprising pectin were responsible for the protection. In a rat model of 30-min ischemia followed by 3-h reperfusion, supplementation with larch arabinogalactan (LAG) over 50 mg/kg/day significantly reduced infarct size. Reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry, and immunoblot analyses showed that intake of LAG blocked the steps involved in apoptotic cascades through downregulation of gelsolin gene expression at the protein (Gelsolin) level, inhibition of p38 phosphorylation in mitogen-activated protein kinase (MAPK) pathways, decreased bax/bcl-2 ratio at the protein (Bax/Bcl-2) level, which was correlated with the ratio at the mRNA level, inhibition of the conversion of Procaspase protein to Caspase-3 protein, and consequently a decrease in apoptotic cells. In addition, the intake of LAG reduced the hif1-α gene expression at the protein (HIF1-α) level. These findings suggest that arabinogalactan is an active component of pectin for reducing myocardial injury by inhibiting apoptosis in postocclusion steps, possibly indicating that arabinogalactan can be developed as a cardioprotectant to prevent myocardial injury.

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling of the precapillary pulmonary arteries, with excessive proliferation of vascular cells. This study was performed to examine the effects of long noncoding RNA CPS1 intronic transcript 1 (CPS1-IT) on PAH in rat models of obstructive sleep apnea (OSA) through regulating interleukin (IL)-1β expression. The OSA models were induced in rats, for determination of the CPS1-IT expression. The binding of CPS1-IT and hypoxia-inducible factor 1 (HIF1) was verified. To analyze the effects of CPS1-IT on PAH, the overexpression vector of CPS1-IT and HIF1, shRNA against IL-1β and pyrrolidine dithiocarbamate (PDTC, inhibitor of the NF-κB signaling pathway) were injected into rat models, respectively. The blood pressure and activity of biochemical indicators including nitric oxide (NO), nitric oxide synthase (NOS), superoxide dismutase (SOD), and lipid peroxide (LPO) were assessed. The expression of IL-1β, HIF1, α-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA), and fibronectin (FN) was determined. The relationship of CPS1-IT to IL-1β and NF-κB was evaluated. CPS1-IT was downregulated in the OSA rat model. Overexpressed CPS1-IT increased the activity of NO, NOS, and SOD as well as α-SMA expression, whereas decreasing LPO activity and expression of PCNA and FN, whereby PAH was suppressed. Notably, overexpressed CPS1-IT reduced IL-1β expression through NF-κB signaling pathway via inhibiting the HIF1 transcriptional activity, suggesting a mechanism affecting PAH. To conclude, overexpressed CPS1-IT alleviated PAH in OSA by reducing IL-1β expression, the mechanism of which was involved with inhibited HIF1 transcriptional activity and the NF-κB signaling pathway.

Myocardial ischemia-reperfusion (I/R) injury is considered to have a detrimental role in coronary heart disease, which is considered to be the leading cause of death worldwide. However, the molecular mechanism involved in the progression of myocardial I/R injury is still unclear. The current study aimed to investigate the expression and function of microRNA (miR)-138 in the process of myocardial I/R injury. First, miR-138 expression levels were analyzed both in myocardium with I/R injury and control myocardium using reverse transcription-quantitative polymerase chain reaction analysis. Then, the relationship between the levels of miR-138 and hypoxia-inducible factor (HIF)1-α was also investigated using a luciferase reporter assay. Assessment of myocardial infarct size, measurements of serum myocardial enzymes and electron microscopy analysis were all utilized to analyse the effect of miR-138 on myocardial I/R injury. The authors of current study also used western blotting to examine the expression levels of the mitochondrial fission-related proteins dynamin-1-like protein and mitochondrial fission 1 protein. It was found that miR-138 is downregulated and HIF1-α is upregulated after myocardial ischemia reperfusion injury. Overexpression of miR-138 reduced myocardial I/R injury-induced infarct sizes and myocardial enzyme levels, and it also inhibited the expression of proteins related to mitochondrial morphology and myocardial I/R-induced mitochondrial apoptosis by targeting HIF1-α. Taken together, these findings provide a novel insight into the molecular mechanism of miR-138 and HIF1-α in the progression of myocardial I/R injury. miR-138 has the potential to become a promising therapeutic target for treating myocardial I/R injury.

Hypoxia-inducible factor-1alpha (HIF-1 alpha) has been reported to have an important role in the metabolism and synthesis of extracellular matrix of the nucleus pulposus cells (NPCs) and was assumed to be involved in the process of intervertebral disc degeneration. The objective of this study was to investigate the role of HIF-1alpha in disc degeneration in vivo using a conditional HIF-1alpha knockout (KO) mouse model. ShhCre transgenic mice were mated with HIF-1 alpha fl/fl mice to generate conditional HIF-1alpha KO mice (HIF-1alpha fl/fl-ShhCre+). Three mice of each genotype (Wide-type and HIF-1alpha KO) at the age of 3 days, 6, and 12 weeks were sacrificed after genotyping. Five lumbar disc samples were harvested from each mouse, with a total of 45 disc samples for each genotype. In situ hybridization and immunohistochemical analysis were used to check the efficacy of HIF-1alpha knockout. Histological grading of the disc degeneration was performed according to the classification system proposed by Boos et al. Picro-sirius red staining, Safranine O/fast green staining and immunohistochemical study were used to evaluate the expression of aggrecan, type-II collagen and vascular endothelial growth factor (VEGF). Histologic analysis revealed more NPC deaths and signs of degeneration in HIF-1alpha KO mice and the degeneration scores of HIF-1alpha KO mice were significantly higher than those of the Wide-type mice at the age of 6 weeks and 12 weeks. There were less expressions of aggrecan, type-II collagen and VEGF in the intervertebral discs of HIF1-alpha KO mice than in those of wild-type mice. Taken together, the results of our study indicated that HIF-1alpha is a pivotal contributor to NPC survival and the homeotasis of extracellular matrix through the HIF-1alpha/VEGF signaling pathway, and plays an important role in the development of disc degeneration.

This study aimed to compare main molecular markers of hypoxia (HIF1-α and P53) and inflammation (TLR-2, TLR-4 and TNF-α) pathways between infertile men with varicocele and fertile individuals. Sperm parameters such as sperm concentration, motility and morphology were assessed according to World Health Organization (Laboratory manual for the examination and processing of human semen. Geneva, Switzerland, 2010) guideline in 20 infertile men with grade II or III varicocele, and 20 fertile men candidate of family balancing. In addition, sperm DNA fragmentation and molecular markers involved in hypoxia and inflammation pathways were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and real-time PCR respectively. Mean of sperm parameters (concentration, motility and morphology) and DNA integrity were significantly lower in infertile men with varicocele compared to fertile individuals. Unlike markers involved in inflammation pathway, mean expression of markers of hypoxia pathway (HIF1-α and P53) was significantly higher in infertile men with varicocele compared to fertile individuals (p < 0.05), and also a significant correlation was observed between expression of HIF1-α and P53 (r = 0.461; p = 0.003). Overall, the result of this study suggests higher likelihood of involvement of hypoxia pathway, in comparison with inflammation pathway, in pathogenesis varicocele associated with male infertility.

Pregnancy complications are associated with abnormal cytotrophoblast differentiation and invasion. Hydrogen peroxide (H2O2) is an important mediator of oxidative ischemia/reperfusion stress in the placenta. Lycium barbarum polysaccharides (LBP) have been demonstrated to counteract oxidative free radicals. The effects of LBP in trophoblast HTR8/SVneo cells injured with H2O2 were examined. A cell counting kit‑8 assay was performed to detect the effect of LBP at different concentrations on the proliferative ability of H2O2 injured trophoblast cells. Flow cytometry was used to determine the levels of reactive oxygen species (ROS), mitochondria membrane potential (MMP) disruption and apoptosis. Superoxide dismutase (SOD) activity and lactate dehydrogenase (LDH) leakage into the supernatant was detected by ultraviolet spectrophotometry. Reverse transcription‑quantitative polymerase chain reaction and western blot analysis were performed to detect the expression of apoptosis‑associated factors, including survivin, hypoxia inducible factor 1‑α (HIF1‑α), Bcl‑2 apoptosis regulator (Bcl‑2), Bcl‑2 associated X apoptosis regulator (Bax). The results revealed that LBP protected the proliferative ability of trophoblast cells injured with H2O2 in a dose‑dependent manner. LBP inhibited the oxidative stress induced by H2O2, by reducing ROS and LDH levels and increasing SOD activity. Additionally, LBP decreased MMP disruption and cell apoptosis induced by H2O2, by increasing the mRNA and protein expression of survivin, HIF1‑α and Bcl‑2 and decreasing Bax expression. Therefore, it was concluded that LBP protected human trophoblast cells from H2O2‑induced oxidative stress and cell apoptosis via regulation of apoptosis‑associated factor expression. It will provide a novel strategy for the treatment of pregnancy complications.

Aerobic glycolysis is a crucial event in fibroblast differentiation, and extracellular matrix (ECM) production in the progression of pulmonary fibrosis (PF). Abnormal high mobility group protein B1 (HMGB1) activation is involved in the pathogenesis of PF. However, whether aerobic glycolysis contributes to HMGB1-induced fibroblast proliferation and ECM production in PF has not yet been determined. In this study, we investigated the effects of HMGB1 on human embryonic lung fibroblast (HLF-1) proliferation, ECM production, and aerobic glycolysis. The lactate dehydrogenase inhibitor oxamic acid (OA), and PFKFB3 inhibitor 3PO were used to block certain crucial steps of aerobic glycolysis. As a result, we observed an increase of HMGB1 in bronchoalveolar lavage fluid (BALF) in bleomycin (BLM)-treated rats as compared to non-treated rats (control group). A concentration-dependent increase of HLF-1 proliferation and expression of α-SMA and α-collagen I were observed in the HMGB1 group, as well as increases of LDHA activation, glucose uptake levels, glycolytic rate, lactate level, and ATP production. OA and 3PO, or suppression of HIF1-α, blocked the effects of HMGB1. In summary, HMGB1 promotes fibroblast proliferation and ECM production though upregulating expression of HIF1-α to induce an increase of aerobic glycolysis.

Hypoxia-inducible factor 1 (HIF1) is a key regulator of angiogenesis and is involved in inflammation, which are two important features of the pathogenesis of peripheral artery disease (PAD). The gene for the HIF1-alpha subunit (HIF1A) carries two common mis-sense mutations, P582S (C>T, rs11549465) and A588T (G>A, rs11549467), which both have been related to increased trans-activation capacity of HIF1-alpha. The aim of the present study was to analyze the role of these polymorphisms in PAD. The study was designed as a case-control study including 917 patients with documented PAD and 969 control subjects. HIF1A genotypes were determined by exonuclease (TaqMan) assays. HIF1A P582S genotype frequencies were not significantly different between PAD patients (PP 82.2%; PS 16.5%; SS 1.3%) and control subjects (83.2%; 15.3%; 1.5%; p = 0.72). Similarly, HIF1A A588T genotype frequencies did not differ significantly between PAD patients (AA 95.9%; AT 4.1%) and control subjects (AA 96.8%; AT 3.2%; p = 0.28). In a multivariate logistic regression analysis including age, sex, smoking, diabetes, arterial hypertension and hypercholesterolemia, neither the HIF1A P582S polymorphism (odds ratio: 1.26; 95% confidence interval 0.92-1.74; p = 0.16) nor the A588T polymorphism (odds ratio: 1.17; 95% confidence interval 0.59-2.35; p = 0.66) was significantly associated with the presence of PAD. Both polymorphisms were furthermore not associated with age at onset of PAD, Fontaine stage of the disease or the ankle/brachial index of patients. We conclude that functional polymorphisms in the HIF1A gene do not contribute to susceptibility to PAD.

BACKGROUND

Electroacupuncture (EA) shows anti-inflammation and several pleiotropic effects that interact with metabolic pathways. In the present study we tested the hypothesis that EA prevents inflammatory response and weight gain in obese mice through modulation of hypoxia-inducible factors-1α (HIF1-α)-dependent pathways in white adipose tissues.

METHODS

Mice were divided in 4 groups: Non-obese, ob/ob, ob/ob submitted to 3 treatments, ob/ob submitted to 7 treatments. Low-frequency EA (2 Hz) was applied at the Zusanli (ST36) acupoint 10 min three times weekly for one or two consecutive weeks in male ob/ob mice. At 22 weeks of age, plasma lipid, glucose, other metabolites and relevant markers were measured by standard assays. Adipose tissue was assessed with immunohistochemical staining. Adipose tissue extracts were also analyzed with quantitative real-time polymerase chain reaction (Q-PCR) and Western blotting.

RESULTS

EA treatment is associated with decreased adipose tissue inflammation, and markedly decreased fat mass and adipocyte size in ob/ob mice. In obese mice, The protein levels of HIF-α were increased, EA shown a marked trend in inhibiting the hypoxic response in adipose tissue. The expression level of hypoxia-related genes (vascular endothelial growth factor A, VEGFA; glucose transporter type 1, Slc2al; glutathione peroxidase 1, GPX1) and inflammation-related genes (TNF-α, IL-6, MCP-1) expression were also reduced in adipose tissue after EA treatment. EA treatment decreased the macrophage recruitment and infiltration (F4/80), and in addition we found that decrease in NF-κB and increase in IkBα were both correlated to reduction in inflammatory processes in adipose tissue. This phenomenon was paralleled by the decrease in the levels of inflammatory cytokines, such as TNF-α, IL-6 and IL-1β in obese mice.

CONCLUSIONS

We conclude that EA prevents weight gain through modulation of HIF-1α-dependent pathways and inflammatory response in obese adipose tissues.

BACKGROUND

Exacerbated proliferation of cancer cells in nascent tumors leads to the genesis of a hypoxic microenvironment, which is associated with poor patient prognosis, because these stress conditions enhance migratory, invasive and metastatic capacities of tumor cells. These changes are associated with the induction of the hypoxia-inducible factors (HIFs, mainly HIF1α) and increased expression of target genes, including Caveolin-1 (CAV1). Results from our group have shown that CAV1 expression in metastatic cancer cells promotes cell migration/invasion in vitro and metastasis in vivo in a manner dependent on tyrosine-14 phosphorylation by src family kinases. Here, we evaluated whether hypoxia-induced expression of CAV1 was required for hypoxia-dependent migration and invasion in cancer cells.

METHODS

B16-F10 murine melanoma and HT29(US) colon adenocarcinoma cells were exposed to hypoxia (1% O2). CAV1 expression was evaluated by western blotting. Endogenous CAV1 and HIF1α were knocked-down using different shRNA constructs. Cell migration and invasion were evaluated in Boyden Chamber and Matrigel assays, respectively.

RESULTS

We observed that hypoxia increased CAV1 protein levels in a HIF1 α- dependent manner, in B16-F10 and HT29(US) cells. Importantly, hypoxia-dependent migration of both tumor cell lines was blocked upon CAV1 knock-down. Likewise, pharmacological inhibition of HIF prevented hypoxia-induced migration and invasion in B16-F10 cells. Finally, hypoxia-induced migration was also blocked by the src-family kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl) pyrazolo3,4-dpyrimidine (PP2), an inhibitor of CAV1 phosphorylation.

CONCLUSIONS

Hypoxia induced migration and invasion of metastatic cancer cells require HIF1α-dependent induction of CAV1 expression and src family kinase activation.

Purpose: Protein levels and activity of the hypoxia-inducible transcription factors HIF-1 and HIF-2 are controlled by hydroxylation of the regulatory alpha chains. Proline hydroxylases (PHDs) target the protein for degradation via the von-Hippel-Lindau (VHL)-ubiquitin-ligase complex, and asparagine hydroxylation by Factor Inhibiting HIF (FIH) leads to transcriptional inactivation. In cell-free systems, these enzymes require ascorbate as a cofactor, and this is also inferred to be an intracellular requirement for effective hydroxylation. However, how intracellular concentrations of ascorbate affect hydroxylase activity is unknown. In this study, we investigated the modulation of the regulatory hydroxylases in cancer cells by intracellular ascorbate. Materials and methods: To facilitate this investigation, we used clear cell renal carcinoma cell lines that were VHL-proficient (Caki-1), with a normal hypoxic response, or VHL-defective (Caki-2 and 786-0), with uncontrolled accumulation of HIF-α chains. We monitored the effect of intracellular ascorbate on the hypoxia-induced accumulation of HIF-1α, HIF-2α and the expression of downstream HIF targets BNIP3, cyclin D1 and GLUT1. Changes in hydroxylation of the HIF-1α protein in response to ascorbate were also investigated in 786-0 cells gene-modified to express full-length HIF-1α (786-HIF1). Results: In VHL-proficient cells, hypoxia induced accumulation of HIF-1α and BNIP3 which was dampened in mild hypoxia by elevated intracellular ascorbate. Increased HIF-2α accumulation occurred only under severe hypoxia and this was not modified by ascorbate availability. In VHL-defective cells, ascorbate supplementation induced additional accumulation of HIF under hypoxic conditions and HIF pathway proteins were unchanged by oxygen supply. In 786-HIF1 cells, levels of hydroxylated HIF-1α were elevated in response to increasing intracellular ascorbate levels. Conclusion: Our data provide evidence that the hypoxic pathway can be modulated by increasing HIF hydroxylase activity via intracellular ascorbate availability. In VHL-defective cells, accumulation of HIF-alpha proteins is independent of hydroxylation and is unaffected by intracellular ascorbate levels.

To investigate the effects of combined therapy with an anticholinergic agent and a β3-adrenoceptor agonist on bladder dysfunction and proliferation-related molecule expression in rats with spinal cord injury (SCI).

The spinal cord was transected at the level of T8-9 in female Sprague-Dawley rats, which were divided into four groups; A: Vehicle, B: 10 mg/kg/day of oxybutynin, C: 10 mg/kg/day of mirabegron, and D: combined administration of oxybutynin and mirabegron. Drugs were administered by oral gavage from 2 to 4 weeks after spinal cord transection. We evaluated urodynamic parameters and bladder tissue remodeling factors.

Non-voiding contractions (NVCs) during the storage phase of cystometrograms tended to be decreased in all three treated groups with a significant reduction in group D versus A. Bladder compliance was improved, and intercontraction intervals, voided volume and bladder capacity were increased in group D. In all three treated groups (B-D), the expression of HIF1-α and TGF-β1 was decreased compared to group A. The expression of collagen-III and bFGF was decreased in groups B and D. The total bladder elastin level was increased in group D.

The combination therapy of an anticholinergic agent and a β3-adrenoceptor agonist elevated the bladder elastin level, reduced NVCs, and increased bladder compliance more effectively than the monotherapy in SCI rats. Thus, the combination therapy could be effective for the treatment of neurogenic bladder dysfunction including bladder remodeling. Neurourol. Urodynam. 36:1039-1045, 2017. © 2016 Wiley Periodicals, Inc.

Triptolide has been demonstrated to induce tumor cell apoptosis. However, the role of triptolide in breast cancer angiogenesis remains unclear. The present study aimed to investigate the function of triptolide in breast cancer and the molecular mechanisms underlying this. The results revealed that triptolide could significantly decrease the expression of vascular endothelial growth factor A (VEGFA) in Hs578T and MDAMB231 breast cancer cells. Furthermore, human umbilical vein endothelial cells were used to perform tube formation and bromodeoxyuridine incorporation assays, which demonstrated an antiangiogenic effect of triptolide. In addition, the effect of triptolide in vivo was examined in a xenograft mouse model, which determined that VEGFA, cluster of differentiation 31 and anti-proliferation marker protein Ki67 expression in tumor sections was decreased in the triptolide treatment group compared with the control group. Western bolt analysis was performed to investigate the phosphorylation of extracellular signal-related kinase (ERK)1/2 and RAC-α serine/threonine-protein kinase after triptolide treatment, and it's effect on hypoxia inducible factor (HIF)1-α expression. The results demonstrated that triptolide suppressed ERK1/2 activation and HIF1-α expression. Furthermore, overexpression of HIF1-α could partially abrogate the inhibitory effect of triptolide on VEGFA expression. These results suggest that triptolide inhibits breast cancer cell angiogenesis in vitro and in vivo through inhibiting the ERK1/2-HIF1-α-VEGFA axis.

[FeFe]-hydrogenase-like genes encode [Fe4 S4]-containing proteins that are ubiquitous in eukaryotic cells. In humans, iron-only hydrogenase-like protein 1 (IOP1) represses hypoxia inducible factor-1α subunit (HIF1-α) at normal atmospheric partial O2 pressure (normoxia, 21 kPa O2). In yeasts, the nar1 mutant cannot grow at 21 kPa O2, but can develop at a lower O2 pressure (2 kPa O2). We show here that plant [FeFe]-hydrogenase-like GOLLUM genes are essential for plant development and cell cycle progression. The mutant phenotypes of these plants are seen in normoxic conditions, but not under conditions of mild hypoxia (5 kPa O2). Transcriptomic and metabolomic experiments showed that the mutation enhances the expression of some hypoxia-induced genes under normal atmospheric O2 conditions and changes the cellular content of metabolites related to energy metabolism. In conclusion, [FeFe]-hydrogenase-like proteins play a central role in eukaryotes including the adaptation of plants to the ambient O2 partial pressure.