BNIP3 mRNA Expression Research Articles

Allantopyrone A interferes with the degradation of hypoxia-inducible factor 1α protein by reducing proteasome activity in human fibrosarcoma HT-1080 cells.

Allantopyrone A is an α-pyrone metabolite that was originally isolated from the endophytic fungus Allantophomopsis lycopodina KS-97. We previously demonstrated that allantopyrone A exhibits anti-cancer, anti-inflammatory, and neuroprotective activities. In the present study, we showed that allantopyrone A up-regulated the protein expression of hypoxia-inducible factor (HIF)-1α in human fibrosarcoma HT-1080 cells. It also up-regulated the mRNA expression of BNIP3 and ENO1, but not other HIF target genes or HIF1A. Allantopyrone A did not inhibit the prolyl hydroxylation of HIF-1α, but enhanced the ubiquitination of cellular proteins. Consistent with this result, chymotrypsin-like and trypsin-like proteasome activities were reduced, but not completely inactivated by allantopyrone A. Allantopyrone A decreased the amount of proteasome catalytic subunits. Therefore, the present results showed that allantopyrone A interfered with the degradation of HIF-1α protein by reducing proteasome activity in human fibrosarcoma HT-1080 cells.

EPOR/CD131-опосредованная ретинопротекция при ротенон-индуцированной нейротоксичности связана с увеличением экспрессии генов аутофагии

Mitochondrial dysfunction is a key driver of neurodegeneration. This study aimed to evaluate the protective potential of EPOR/CD131 (heterodimeric erythropoietin receptor) stimulation in the neurodegeneration caused by rotenone-induced mitochondrial dysfunction. The effects of erythropoietin (EPO) and an EPO mimetic peptide pHBSP were assessed using in vivo and in vitro models. Single injections of 10 µg/kg EPO or 5 µg/kg pHBSP significantly alleviated the degeneration of ganglion cells of the retina in a rotenone-induced retinopathy in rats (p < 0.05). Consistently, in vitro exposure of rotenone-treated murine primary neuroglial cultures to 500 nM EPO or pHBSP significantly rescued the survival of the cells (p < 0.005). The observed enhancement of LC3A, ATG7, Beclin-1, Parkin and BNIP3 mRNA expression by EPOR/CD131 agonists implicates the autophagy and mitophagy activation as a plausible mitoprotective mechanism.

C1 Esterase Inhibitor Reduces BBB Leakage and Apoptosis in the Hypoxic Developing Mouse Brain

Inflammatory pathways involved in blood-brain barrier (BBB) vulnerability and hypoxic brain oedema in models of perinatal brain injury seem to provide putative therapeutic targets. To investigate impacts of C1-esterase inhibitor (C1-INH; 7.5-30IU/kg, i.p.) on functional BBB properties in the hypoxic developing mouse brain (P7; 8% O2 for 6h), expression of pro-apoptotic genes (BNIP3, DUSP1), inflammatory markers (IL-1ß, TNF-alpha, IL-6, MMP), and tight junction proteins (ZO-1, occludin, claudin-1, -5), and S100b protein concentrations were analysed after a regeneration period of 24h. Apoptotic cell death was quantified by CC3 immunohistochemistry and TUNEL staining. In addition to increased apoptosis in the parietal cortex, hippocampus, and subventricular zone, hypoxia significantly enhanced the brain-to-plasma albumin ratio, the cerebral S100b protein levels, BNIP3 and DUSP1 mRNA concentrations as well as mRNA expression of pro-inflammatory cytokines (IL-1ß, TNF-alpha). In response to C1-INH, albumin ratio and S100b concentrations were similar to those of controls. However, the mRNA expression of BNIP3 and DUSP1 and pro-inflammatory cytokines as well as the degree of apoptosis were significantly decreased compared to non-treated controls. In addition, occludin mRNA levels were elevated in response to C1-INH (p < 0.01). Here, we demonstrate for the first time that C1-INH significantly decreased hypoxia-induced BBB leakage and apoptosis in the developing mouse brain, indicating its significance as a promising target for neuroprotective therapy.

Hyperglycemia differentially affects proliferation, apoptosis, and BNIP3 and p53 mRNA expression of human umbilical cord Wharton's jelly cells from non-diabetic and diabetic pregnancies

Diabetes in pregnancy constitutes an unfavorable environment for embryonic and fetal development, where the child has a higher risk of perinatal morbidity and mortality, with high incidence of congenital malformations and predisposition to long-term metabolic diseases that increase with a hypercaloric diet. To analyze whether hyperglycemia differentially affects proliferation, apoptosis, and mRNA expression in cells from children of normoglycemic pregnancies (NGPs) and diabetes mellitus pregnancies (DMPs), we used umbilical cord Wharton jelly cells as a research model. Proliferation assays were performed to analyze growth and determine the doubling time, and the rate of apoptosis was determined by flow cytometry-annexin-V assays. AMPK, BNIP3, HIF1α, and p53 mRNA gene expression was assessed by semi-quantitative RT-PCR. We found that hyperglycemia decreased proliferation in a statistically significant manner in NGP cells treated with 40 mM D-glucose and in DMP cells treated with 30 and 40 mM D-glucose. Apoptosis increased in hyperglycemic conditions in NGP and DMP cells. mRNA expression of BNIP3 and p53 was significantly increased in cells from DMPs but not in cells from NGPs. We found evidence that maternal irregular metabolic conditions, like diabetes with hyperglycemia in culture, affect biological properties of fetal cells. These observations could be a constituent of fetal programming.

OR-024 Changes of mitochondrial autophagy - related genes and autophagosome after skeletal muscle blunt trauma

Objective Objective: To study the changes of mitochondrial autophagy-related genes and autophagosome after skeletal muscle blunt trauma, to reveal the changes of mitochondrial adaptive repair process after skeletal muscle blunt trauma, and to elucidate the mechanism of blunt trauma repair process.&#x0D; Methods Methods: Sixty - four male Wistar rats were randomly divided into control group and blunt trauma group三 (divided into 12h group, 2d group, 5d group, 7d group, 10d group, 15d group and 30d group) according to the time of extraction. The expression of HIF-1α, AMPKα2, BNIP3 and NIX protein in skeletal muscle hypoxia and autophagy-related factors were measured by Western-Blot. QRT-PCR was performed to analyze the expression levels of HIF-1α, AMPKα2, BNIP3 and NIX. The ultrastructure and autophagic formation at different time points were observed by transmission electron microscopy (TEM).&#x0D; Results Results: The expression of HIF-1α and AMPKα2 protein reached the peak at 12h and 2d, and the expression of HIF-1α was significantly higher than that of the control group (P &lt;0.05). The expression of AMPKα2 was significantly higher at 5 days after injury (P &lt;0.05), and reached the normal level at 10 days. BNIP3 began to decline after 5 days, but still higher than normal at 10 days after treatment. NIX expression peak appeared at 12h and 2d after injury, with high-express to 7d. The expression of HIF-1α and AMPKα2 mRNA was significantly higher than that of the control group (P &lt;0.01), but decreased until 5d (P &lt;0.05), then decreased to normal level. The mRNA expression of BNIP3 and NIX was basically the same as their protein performance. A number of autophagosomes were observed at 12 h after injury, and the number of autophagosomes increased gradually at 2-7 d. After 10 days, the number of autophagosomes decreased compared with that of 12 h-7 d after blunt. And after 15 days, the number of autophagosites decreased gradually.&#x0D; Conclusions Conclusion: The changes of early stage metabolic regulator AMPKα2 and hypoxia-sensitive factor HIF-1αafter skeletal muscle blunt trauma indicated that an energy crisis occurred in the skeletal muscle after injury, and the hypoxic environment was formed. The mitochondrial autophagy, the expression of BNIP3 and NIX showed that mitochondrial autophagy was activated and hypoxia induced mitochondrial autophagy at early skeletal muscle contusion peroid. Hypoxia-induced mitochondrial autophagy could remove the damaged mitochondria, maintain mitochondrial quality and provide raw materials for new mitochondria generation, facilitate the rapid recovery of damaged skeletal muscle, which may be a compensatory mechanism of the body response to injury.

Hyperoside protects against hypoxia/reoxygenation induced injury in cardiomyocytes by suppressing the Bnip3 expression

AimsRole of hyperoside in protecting cardiomyocytes from ischemia/reperfusion induced injury has been proved. However, possible protecting mechanisms remain unclear. To fix the problem, an essential pro-apoptotic protein Bnip3 was studied in our experiments. Methods and resultsNeonatal rat cardiomyocytes were used and submitted to hypoxia for 8h followed by reoxygenation for 2h to simulate the ischemia/reperfusion injury. Hypoxia/reoxygenation(H/R) induced damage to cardiomyocytes and the protective effect of hyperoside were examined by means of MTT assay. H/R-induced apoptosis was assessed by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL) and DNA Ladder assay. mRNA expression of Bnip3 was determined by use of quantitative real-time reverse transcription polymerase chain reaction assay. Protein levels of Bnip3, Bax, Bcl-2 and cleaved caspase-3 were examined using western-blot assay. Our results showed that H/R caused great damage to cardiomyocytes, upregulated the protein expressions of Bnip3, Bax, cleaved caspase3, and decreased the expression of the anti-apoptotic protein of Bcl-2. Whereas, compared with the H/R group, a decrease in activities of Bnip3, Bax, cleaved caspase3, and a promoting expression of Bcl-2 were detected in the H/R goup pretreated with hyperoside. ConclusionIt was concluded in our study that H/R-induced apoptotic effect in cardiomyocytes could be attenuated by hyperoside, and the protective role of hyperoside, if not completely, could be partly through the suppression of the pro-apoptotic gene Bnip3.

Autophagy Induction Results in Enhanced Anoikis Resistance in Models of Peritoneal Disease.

Development of BNIP3/BNIP3L-targeting agents or autophagy-targeting agents may reduce morbidity and mortality associated with peritoneal carcinomatosis and sarcomatosis. Mol Cancer Res; 15(1); 26-34. ©2016 AACR.

Acute environmental hypoxia activates autophagy in human skeletal muscle (1167.2)

Hypoxia‐induced muscle wasting is a phenomenon often described at high altitude, which has been attributed to an altered protein metabolism. We hypothesized that acute normobaric hypoxia would favour a negative net protein balance by repressing anabolic and activating proteolytic signaling pathways at rest and post‐exercise. Twenty‐two subjects participated in an experimental trial in normoxia and hypoxia (10.7% O2). Muscle biopsies were obtained before and after a 20‐min moderate exercise. In hypoxia, autophagic flux was increased as indicated by an increased LC3‐II/I ratio and LC3‐II expression and decreased p62/SQSTM1 expression. Following exercise in hypoxia a decrease in LC3‐I, LC3‐II and LC3‐II/I as well as in p62/SQSTM1 expressions was found. Bnip3 mRNA expression, a marker of mitophagy, was also increased in hypoxia. MAFbx mRNA expression, a muscle‐specific E3‐ligase, was increased in hypoxia and returned to levels similar to normoxia after exercise, while MuRF‐1 mRNA was not altered. In hypoxia, REDD1 expression and AMPK phosphorylation, two potential inhibitors of the PKB/mTOR pathway, were increased although phosphorylation of PKB, S6K1 and 4E‐BP1 was not modified. Our results indicate that environmental hypoxia modulates protein metabolism at rest and after moderate exercise by primarily altering protein breakdown, and more specifically the autophagy‐lysosomal system.

BNIP3 Is Downregulated in Myelodysplastic Syndromes and Acute Myeloid Leukemia and Is a Potential Molecular Target for Decitabine

AbstractAbstract 1708In myelodysplastic syndromes (MDS), changes in the balance between pro- and anti-apoptotic proteins are associated with disease progression and evolution towards acute myeloid leukemia (AML). BNIP3 is a pro-apoptotic protein, member of the Bcl-2 superfamily, associated with the pathogenesis of many diseases, including cancer. More recently, BNIP3 was identified as a potent inducer of autophagy. In primary leukemia cells, BNIP3 expression has been shown to be reduced due to epigenetic modifications; however, there is a lack of studies of BNIP3 in MDS. The aim of this study was to characterize BNIP3 mRNA expression levels and DNA methylation status of its 5’ CpG island in bone marrow cells from MDS and AML patients, healthy donors and in leukemia hematopoietic cell lines. We also investigated the effects of BNIP3 silencing upon decitabine (DAC) treatment in U937 cells regarding colony formation, apoptosis and autophagy. Bone marrow aspirates were obtained from 21 healthy donors, 55 patients with MDS and 44 patients with AML at diagnosis. MDS patients were grouped into low-risk and high-risk groups according to FAB (RA/RARS=30; RAEB/RAEBt=25), WHO (RCUD/RCMD/RARS/del5/=29; RAEB1/RAEB2=17) and IPSS (low/INT-1=38; INT2/high=16). The study was approved by the ethics committee and informed written consent was provided. Gene expression was evaluated by qPCR and DNA methylation was performed by Combined Bisulfite Restriction Analysis. BNIP3 knockdown was performed in U937 cells with specific shRNA-expressing lentiviral vector. ShRNA encoding no specific sequence was used as control. Colony formation was carried out in semisolid methylcellulose medium. Apoptosis and autophagy were accessed by flow cytometry. All assays were performed in lentiviral transduced cells treated or not with 5μM DAC for 72 hours. Mann-Whitney and Student’s t-test were used for statistical analyses. We observed a significant decrease in BNIP3 mRNA expression in total bone marrow cells from AML and MDS compared to healthy donors (AML= 0.52 [5.27-0.00]; MDS=0.52 [5.25-0.02] versus healthy donors=1.09 [6.04-0.18]; P<0.05). BNIP3 expression did not differ between low-risk and high-risk MDS patients according to FAB and WHO classifications, and IPSS. Methylation of BNIP3 promoter was detected in 19% (4/21; 2/14 low-risk and 2/7 high-risk) MDS and 26% (4/15) AML patients, but not in any of the 6 healthy donors evaluated. BNIP3 expression was detected in all myeloid cell lines studied (K562, HL60, U937 and P39 cells). The lowest expression was found in U937, which presented methylation of BNIP3 promoter. Interestingly, in U937 cells, DAC treatment resulted in upregulation of BNIP3 expression (3.7-fold), total inhibition of colony formation, increased apoptosis and autophagy (50% for both). The effect of DAC on cell apoptosis was reduced by BNIP3 silencing; apoptosis was reduced by 48%±4.3 in shBNIP3 cells compared to 59%±5.7 in shControl cells (p<0.05). Autophagy was not modulated by DAC treatment in BNIP3 silenced cells. In bone marrow cells from MDS and AML patients, the low frequency of methylated BNIP3 promoter suggests that DNA methylation is not the single reason for the decreased BNIP3 expression in these patients. Other mechanisms, such as histone deacetylation, may be involved in aberrant BNIP3 expression. DAC treatment in BNIP3 silenced cells had no effect on autophagy, suggesting that other mechanisms besides increasing BNIP3 expression is involved in this cell function. On the other hand, DAC treatment in U937 cells was able to induce apoptosis, especially in cells expressing BNIP3, supporting the hypothesis that BNIP3 methylation is a molecular target for DAC. Disclosures:No relevant conflicts of interest to declare.

Differential expression of BNIP family members of BH3-only proteins during the development and after axotomy in the rat.

The BNIPs (BCL2 and adenovirus E1B 19 kDa interacting proteins) are a subfamily of BCL2 family proteins typically containing a single BCL2 homology 3 (BH3) domain. BNIPs exert important roles in two major degradation processes in cells - apoptosis and autophagy. Although it is known that the function of BNIPs is transcriptionally regulated under hypoxic conditions in tumors, their regulation in the developing brain and neurons following the induction of apoptosis/autophagy is largely unknown. In this study, we demonstrate that three members of the BNIP family, BNIP1, BNIP3 and BNIP3L, are expressed in the developing brain with distinct brain region specificity. BNIP3 mRNA was especially enriched in the entorhinal cortex, raising a possibility that it may have additional biological functions in addition to its apoptotic and autophagic functions. Following starvation-induced autophagy induction, BNIP1 mRNA was selectively increased in cultured neurons. However, the apoptogenic chemical staurosporine failed to modulate the expression of BNIPs, which is in contrast to the marked induction of all BNIPs by glucose-oxygen deprivation. Finally, neonatal nerve axotomy, which triggers apoptosis in motoneurons, selectively enhanced BNIP3 mRNA expression. Collectively, these results suggest that the expression of BNIPs is differentially regulated depending on the stimuli, and BNIPs may exert unique biological functions.

Glucocorticoids exacerbate hypoxia-induced expression of the pro-apoptotic gene Bnip3 in the developing cortex

Neonatal administration of the synthetic glucocorticoid, dexamethasone (DEX) retards brain growth, alters adult behaviors and induces cell death in the rat brain, thereby implicating glucocorticoids as developmentally neuroendangering compounds. Glucocorticoids also increase expression of pro-apoptotic Bcl-2 family members and exacerbate expression of hypoxic responsive genes. Bnip3 is a pro-apoptotic Bcl-2 family member that is upregulated in response to hypoxia. In these studies, we investigated the interactions of glucocorticoid receptor and hypoxia in the regulation of Bnip3 mRNA in cortical neurons. Using quantitative real time reverse transcription–polymerase chain reaction, we found that DEX treatment of postnatal days 4–6 rat pups caused a significant increase in Bnip3 mRNA expression compared with vehicle controls. A significant increase in Bnip3 mRNA was also measured in primary cortical neurons 72 h after treatment with RU28362, a glucocorticoid receptor selective agonist. In primary cortical neurons, hypoxia increased Bnip3 mRNA expression and this was exacerbated with RU28362 treatment. To elucidate the mechanism of glucocorticoid- and hypoxia-mediated regulation of Bnip3 transcription, a Bnip3 promoter–luciferase reporter construct was utilized in primary cortical neurons. Upregulation of the Bnip3 promoter was mediated by a single glucocorticoid response element and a hypoxic response element. Bnip3 overexpression in primary cortical neurons significantly increased cell death, which is dependent on the Bnip3 transmembrane domain. However, despite the increased expression of Bnip3 following glucocorticoid and hypoxia treatment, corresponding decreases in cell survival were minimal. These studies identify a novel pathway in the developing cortex through which glucocorticoids may enhance a metabolic insult, such as hypoxia.

Localization and developmental ontogeny of the pro-apoptotic Bnip3 mRNA in the postnatal rat cortex and hippocampus

Naturally occurring cell death occurs during the first two postnatal weeks in the rat cortex and hippocampus. During this process, apoptosis is initiated by activating or altering expression of pro-apoptotic members of the Bcl-2 family. Bnip3 is a pro-apoptotic member of the Bcl-2 family that induces cell death by opening the mitochondrial permeability transition pore. To date, Bnip3 expression in the central nervous system has only been examined during hypoxia-mediated apoptosis in the adult rat brain. In this study, we investigated the localization and ontogeny of Bnip3 mRNA expression in the postnatal male and female rat brain. Bnip3 mRNA was localized by in situ hybridization in the neonatal cortex, hippocampus, habenula and thalamus. Using quantitative real-time RT-PCR, Bnip3 mRNA levels were found to be greatest at postnatal day 6.5 in the female anterior and posterior cingulate cortices and hippocampus. Bnip3 mRNA expression also increased in the male anterior cingulate cortex at postnatal day 6.5. However, a developmental change in Bnip3 levels did not occur in the male posterior cingulate cortex and hippocampus. In the anterior cingulate cortex on postnatal day 6.0 and adulthood, female rats had significantly greater levels of Bnip3 mRNA compared to that of males. Altering levels of testosterone in the neonatal rat did not alter the sex differences in Bnip3 mRNA levels. The transient increase in Bnip3 mRNA expression correlates with naturally occurring cell death in the neonatal rat cortex and hippocampus. Thus, Bnip3 may be a mediator of developmental apoptosis in the postnatal rat brain.

Hypoxia and expression of the proapoptotic regulator BNIP3 in cervical cancer

Hypoxia plays a major role in the malignant progression of tumors. Here, we investigate the expression of Bcl-2/adenovirus E1B 19 kd-interacting protein 3 (BNIP3), a proapoptotic Bcl-2 family member, and its relationship to hypoxia in cervical cancer cell lines and clinical samples of cervical cancer. Cervical cancer cell lines were grown under hypoxia or normoxia, and BNIP3 mRNA expression was examined by Northern blot analysis. In 50 patients with cervical cancer, intratumoral oxygen measurement with the Eppendorf electrode and needle biopsies of the tumor were performed. The obtained tissue was subsequently analyzed by immunohistochemistry with an anti-BNIP3 antibody. Cervical cancer tissue collected upon surgery was used for Northern blot analysis ofin vivoBNIP3 mRNA expression. BNIP3 mRNA is strongly induced under hypoxic conditions in all cervical cancer cell lines investigated. Furthermore, Northern blot analysis revealed that BNIP3 mRNA is expressed in cervical cancer tissue. Using immunohistochemistry, we demonstrated that BNIP3 protein is expressed in 82% of the investigated cervical cancers and that more advanced tumor stages showed significantly stronger BNIP3 expression. However, we observed no correlation between BNIP3 expression and intratumoral hypoxia. In conclusion, BNIP3 is expressed in different cervical cancer cell lines as well as in clinical samples of cervical cancer. Although BNIP3 is clearly hypoxia-induciblein vitro, our results suggest additional mechanisms of BNIP3 regulationin vivo.Our findings therefore highlight a discrepancy betweenin vitromodels of tumor hypoxia and the complexity of human cancer.

Hypoxia and expression of the proapoptotic regulator BNIP3 in cervical cancer

Hypoxia plays a major role in the malignant progression of tumors. Here, we investigate the expression of Bcl-2/adenovirus E1B 19 kd-interacting protein 3 (BNIP3), a proapoptotic Bcl-2 family member, and its relationship to hypoxia in cervical cancer cell lines and clinical samples of cervical cancer. Cervical cancer cell lines were grown under hypoxia or normoxia, and BNIP3 mRNA expression was examined by Northern blot analysis. In 50 patients with cervical cancer, intratumoral oxygen measurement with the Eppendorf electrode and needle biopsies of the tumor were performed. The obtained tissue was subsequently analyzed by immunohistochemistry with an anti-BNIP3 antibody. Cervical cancer tissue collected upon surgery was used for Northern blot analysis of in vivo BNIP3 mRNA expression. BNIP3 mRNA is strongly induced under hypoxic conditions in all cervical cancer cell lines investigated. Furthermore, Northern blot analysis revealed that BNIP3 mRNA is expressed in cervical cancer tissue. Using immunohistochemistry, we demonstrated that BNIP3 protein is expressed in 82% of the investigated cervical cancers and that more advanced tumor stages showed significantly stronger BNIP3 expression. However, we observed no correlation between BNIP3 expression and intratumoral hypoxia. In conclusion, BNIP3 is expressed in different cervical cancer cell lines as well as in clinical samples of cervical cancer. Although BNIP3 is clearly hypoxia-inducible in vitro, our results suggest additional mechanisms of BNIP3 regulation in vivo. Our findings therefore highlight a discrepancy between in vitro models of tumor hypoxia and the complexity of human cancer.