BDH1-mediated ßOHB metabolism ameliorates diabetic kidney disease by activation of NRF2-mediated antioxidative pathway.

A ketogenic diet (KD) and ß-hydroxybutyrate (ßOHB) have been widely reported as effective therapies for metabolic diseases. ß-Hydroxybutyrate dehydrogenase 1 (BDH1) is the rate-limiting enzyme in ketone metabolism. In this study, we examined the BDH1-mediated ßOHB metabolic pathway in the pathogenesis of diabetic kidney disease (DKD). We found that BDH1 is downregulated in the kidneys in DKD mouse models, patients with diabetes, and high glucose- or palmitic acid-induced human renal tubular epithelial (HK-2) cells. BDH1 overexpression or ßOHB treatment protects HK-2 cells from glucotoxicity and lipotoxicity by inhibiting reactive oxygen species overproduction. Mechanistically, BDH1-mediated ßOHB metabolism activates NRF2 by enhancing the metabolic flux of ßOHB-acetoacetate-succinate-fumarate. Moreover, in vivo studies showed that adeno-associated virus 9-mediated BDH1 renal expression successfully reverses fibrosis, inflammation, and apoptosis in the kidneys of C57 BKS db/db mice. Either ßOHB supplementation or KD feeding could elevate the renal expression of BDH1 and reverse the progression of DKD. Our results revealed a BDH1-mediated molecular mechanism in the pathogenesis of DKD and identified BDH1 as a potential therapeutic target for DKD.

D-ß-Hydroxybutyrate Dehydrogenase Mitigates Diabetes-Induced Atherosclerosis through the Activation of Nrf2.

BACKGROUND: We aimed to investigate the role and mechanism of ß-hydroxybutyrate dehydrogenase 1 (Bdh1) in regulating macrophage oxidative stress in diabetes-induced atherosclerosis (AS). METHODS: We performed immunohistochemical analysis of femoral artery sections to determine differences in Bdh1 expression between normal participants, AS patients, and patients with diabetes-induced AS. Diabetic Apoe-/- mice and high-glucose (HG)-treated Raw264.7 macrophages were used to replicate the diabetes-induced AS model. The role of Bdh1 in this disease model was determined by adeno-associated virus (AAV)-mediated overexpression of Bdh1 or overexpression or silencing of Bdh1. RESULTS: We observed reduced expression of Bdh1 in patients with diabetes-induced AS, HG-treated macrophages, and diabetic Apoe-/- mice. AAV-mediated Bdh1 overexpression attenuated aortic plaque formation in diabetic Apoe-/- mice. Silencing of Bdh1 resulted in increased reactive oxygen species (ROS) production and an inflammatory response in macrophages, which were reversed by the ROS scavenger N-acetylcysteine. Overexpression of Bdh1 protected Raw264.7 cells from HG-induced cytotoxicity by inhibiting ROS overproduction. In addition, Bdh1 induced oxidative stress through nuclear factor erythroid-related factor 2 (Nrf2) activation by fumarate acid. CONCLUSION: Bdh1 attenuates AS in Apoe-/- mice with type 2 diabetes, accelerates lipid degradation, and reduces lipid levels by promoting ketone body metabolism. Moreover, it activates the Nrf2 pathway of Raw264.7 by regulating the metabolic flux of fumarate, which inhibits oxidative stress and leads to a decrease in ROS and inflammatory factor production.

PTEN-knockout regulates metabolic rewiring and epigenetic reprogramming in prostate cancer and chemoprevention by triterpenoid ursolic acid.

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is one of the most frequently mutated/deleted tumor suppressor genes in many human cancers. Ursolic acid (UA) is a natural triterpenoid possessing antioxidant, anti-inflammatory, and anticancer effects. However, how PTEN impacts metabolic rewiring and how UA modifies PTEN-driven metabolic and epigenetic reprogramming in prostate cancer (PCa) remains unknown. In the current study, we found that UA protects against PTEN knockout (KO)-induced tumorigenesis at different stages of PCa. Epigenomic CpG methyl-seq revealed UA attenuated PTEN KO-induced differentially methylated regions (DMRs) profiles. Transcriptomic RNA-seq showed UA abrogated PTEN KO-induced differentially expressed genes (DEGs) of PCa-related oncogenes' Has3, Cfh, and Msx1 overexpression, indicating UA plays a crucial role in PTEN KO-mediated gene regulation and its potential consequences on cancer interception. Association analysis of DEGs and DMRs identified that the mRNA expression of tumor suppressor gene BDH2, and oncogenes Ephas, Isg15, and Nos2 were correlated with the promoter CpG methylation status in the early-stage comparison groups indicating UA could regulate the oncogenes or tumor suppressor genes by modulating their promoter methylation at an early stage of prostate tumorigenesis. The metabolomic study showed UA attenuated PTEN KO-regulated cancer-associated metabolisms like purine metabolism/metabolites correlating with RNAseq findings, glycolysis/gluconeogenesis metabolism, as well as epigenetic-related metabolites pyruvate and lactate indicating UA plays a critical role in PTEN KO-mediated metabolic and epigenetic reprogramming and its consequences on cancer development. In this context, UA impacts metabolic rewiring causing epigenetic and transcriptomic reprogramming potentially contributing to the overall protection against prostate-specific PTEN KO-mediated PCa.

The sodium-glucose co-transporter-2 inhibitor ertugliflozin modifies the signature of cardiac substrate metabolism and reduces cardiac mTOR signalling, endoplasmic reticulum stress and apoptosis.

AIM: To investigate cardiac signalling pathways connecting substrate utilization with left ventricular remodelling in a murine pressure overload model. METHODS: Cardiac hypertrophy was induced by transverse aortic constriction surgery in 20-week-old C57BL/6J mice treated with or without the sodium-glucose co-transporter 2 (SGLT2) inhibitor ertugliflozin (225 mg kg-1 chow diet) for 10 weeks. RESULTS: Ertugliflozin improved left ventricular function and reduced myocardial fibrosis. This occurred simultaneously with a fasting-like response characterized by improved glucose tolerance and increased ketone body concentrations. While cardiac insulin signalling was reduced in response to SGLT2 inhibition, AMP-activated protein kinase (AMPK) signalling was increased with induction of the fatty acid transporter cluster of differentiation 36 and phosphorylation of acetyl-CoA carboxylase (ACC). Further, enzymes responsible for ketone body catabolism (ß-hydroxybutyrate dehydrogenase, succinyl-CoA:3-oxoacid-CoA transferase and acetyl-CoA acetyltransferase 1) were induced by SGLT2 inhibition. Ertugliflozin led to more cardiac abundance of fatty acids, tricarboxylic acid cycle metabolites and ATP. Downstream mechanistic target of rapamycin (mTOR) pathway, relevant for protein synthesis, cardiac hypertrophy and adverse cardiac remodelling, was reduced by SGLT2 inhibition, with alleviation of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) providing a potential mechanism for abundant reduced left ventricular apoptosis and fibrosis. CONCLUSION: SGLT2 inhibition reduced left ventricular fibrosis in a murine model of cardiac hypertrophy. Mechanistically, this was associated with reduced cardiac insulin and increased AMPK signalling as a potential mechanism for less cardiac mTOR activation with alleviation of downstream ER stress, UPR and apoptosis.

Labile iron accumulation augments T follicular helper cell differentiation.

T follicular helper (Tfh) cells are a subset of CD4+ T cells that are essential in the pathogenesis of systemic lupus erythematosus (SLE). Notably, iron is required for activated CD4+ T lymphocytes to sustain high proliferation and metabolism. In this issue of the JCI, Gao et al. showed that CD4+ T cells from patients with SLE accumulated iron, augmenting their differentiation into Tfh cells and correlating with disease activity. Using human cells and murine models, the authors demonstrated that miR-21 was overexpressed in lupus T cells and inhibited 3-hydroxybutyrate dehydrogenase-2 (BDH2). The subsequent loss of BDH2 drove labile iron to accumulate in the cytoplasm and promoted TET enzyme activity, BCL6 gene demethylation, and Tfh cell differentiation. This work identifies a role for iron in CD4+ T cell biology and the development of pathogenic effectors in SLE. We await future investigations that could determine whether modulating iron levels could regulate Tfh cells in human health and disease.

BDH2 promotes apoptosis and autophagy of lung adenocarcinoma cells via Akt/mTOR pathway.

Cytoprotective autophagy induces tumor cell apoptosis or autophagic programmed cell death. Autophagy and apoptosis are implicated in the pathogenesis of lung cancer, especially lung adenocarcinoma. 3-Hydroxybutyrate dehydrogenase type 2 (BDH2), a rate-limiting catalyzer in the regulation of intracellular iron metabolism and siderophore biogenesis, has been shown to be a tumor suppressor through promotion of cell apoptosis and autophagy. However, the biological role of BDH2 on lung adenocarcinoma cell apoptosis and autophagy remains unclear. Data from Western blot and qRT-PCR showed that BDH2 was down-regulated in lung adenocarcinoma cells (A549, NCI-H1975, PC9) compared to normal human lung cells (BEAS-2B). Functional assays demonstrated that pcDNA-mediated over-expression of BDH2 reduced cell viability of lung adenocarcinoma cells, and repressed the proliferation. Cell apoptosis of lung adenocarcinoma was promoted by BDH2 over-expression with up-regulation of Bax and cleaved caspase-3. Over-expression of BDH2 reduced protein expression of p62 in lung adenocarcinoma cells, enhanced LC3 and Beclin-1. Phosphorylation of AKT and mTOR in lung adenocarcinoma cells were reduced by BDH2 over-expression. In conclusion, BDH2 functioned as a tumor suppressor in lung adenocarcinoma through promotion of Akt/mTOR-mediated cell apoptosis and autophagy.

System analysis of FHIT in LUAD and LUSC: The expression, prognosis, gene regulation network, and regulation targets.

BACKGROUND: Fragile histidine triad (FHIT) is a strong tumor suppressor gene, and cells deficient in FHIT are prone to acquiring cancer-promoting mutations. Due to its location, deletions within FHIT are common in cancer. Over 50% of cancers show loss of FHIT expression. However, to date, expression levels, gene regulatory networks, prognostic value, and target prediction of FHIT in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) have not been fully reported. Therefore, systematic analysis of FHIT expression, gene regulatory network, prognostic value, and targeted prediction in patients with LUAD and LUSC has important guiding significance, providing new therapeutic targets and strategies for clinical treatment of lung cancer to further improve the therapeutic effect of lung cancer. METHODS: Multiple free online databases were used for the abovementioned analysis in this study, including cBioPortal, TRRUST, Human Protein Atlas, GeneMANIA, GEPIA, Metascape, UALCAN, LinkedOmics, and TIMER. RESULTS: FHIT was upregulated in patients with LUAD, and downregulated in patients with LUSC. Genetic alterations of FHIT were found in patients with LUAD (7%), and LUSC (10%). The promoter methylation of FHIT was lower in patients with LUAD and LUSC. FHIT expression significantly correlated with LUSC pathological stages. Furthermore, patients with LUAD and LUSC having low FHIT expression levels had a longer survival than those having high FHIT expression levels. FHIT and its neighboring genes (the 50 most frequently altered neighboring genes of FHIT) functioned in the regulation of protein kinase and DNA binding in patients with LUAD, as well as cell channels and membrane potential in patients with LUSC. Gene ontology enrichment analysis revealed that the functions of FHIT and its neighboring genes are mainly related to disordered domain-specific binding, protein kinase binding, and ion gated channel activity in patients with LUAD, as well as calcium ion binding and intracellular ligand-gated ion channel activity in patients with LUSC. Transcription factor targets of FHIT and its neighboring genes in patients with lung cancer were found: USF1, SOX6, USF2, SIRT1, VHL, LEF1, EZH2, TP53, HDAC1, ESR1, EGR1, YY1, MYC, RELA, NFKB1, and E2F1 in LUAD; and HDAC1, DNMT1, and E2F1 in LUSC. We further explored the FHIT-associated kinase (PRKCQ, AURKB and ATM in LUAD as well as PLK3 in LUSC) and FHIT-associated miRNA targets (MIR-188, MIR-323, and MIR-518A-2 in LUAD). Furthermore, the following genes had the strongest correlation with FHIT expression in patients with lung cancer: NICN1, HEMK1, and BDH2 in LUAD, and ZMAT1, TTC21A, and NICN1 in LUSC. FHIT expression was positively associated with immune cell infiltration (B cell) in patients with LUAD, as well as B cell, CD8 + T, CD4 + T cells, macrophages, and dendritic cells in patients with LUSC. Nevertheless, FHIT expression was negatively associated with CD8 + T cells and neutrophils in patients with LUAD. CONCLUSIONS: The expression, gene regulatory network, prognostic value and targeted prediction of FHIT in patients with LUAD and LUSC were systematically analyzed and revealed in this study, thereby laying a foundation for further research on the role of FHIT in LUAD and LUSC occurrence. This study provides new LUAD and LUSC therapeutic targets and prognostic biomarkers as a reference for fundamental and clinical research.

RAB27B inhibits proliferation and promotes apoptosis of leukemic cells via 3-Hydroxy butyrate dehydrogenase 2 (BDH2).

RAB27B is a member of Ras-like small GTPases that plays a role in endocytosis, exocytosis, and vesicle trafficking. We made an attempt to study the impacts of RAB27B on the proliferation and apoptosis of acute myeloid leukemia (AML) cells. The silencing of RAB27B was induced by siRNA for the detection of proliferation, cell cycle, and apoptosis, respectively by Cell Counting Kit-8 (CCK8), flow cytometry, and TUNEL. Related markers were also evaluated by Western blot analysis. The interaction between RAB27B and BDH2 was predicted by bioinformatics analysis and determined by immunoprecipitation. The gain of function of BDH2 was also detected by these functional assays. RAB27B exhibited high levels in AML cells, and RAB27B silencing led to reduced proliferation, increased cell cycle arrest and apoptosis levels. Then, the interaction between RAB27B and BDH2 was confirmed. Moreover, the effects of RAB27B inhibition on the proliferation, cell cycle arrest, and cell apoptosis were abolished after BDH2 overexpression. RAB27B inhibits proliferation and promotes apoptosis of leukemic cells by interacting with BDH2. Targeting RAB27B might be an effective method for the treatment of AML.

BDH2 triggers ROS-induced cell death and autophagy by promoting Nrf2 ubiquitination in gastric cancer.

BACKGROUND: 3-Hydroxy butyrate dehydrogenase 2 (BDH2) is a short-chain dehydrogenase/reductase family member that plays a key role in the development and pathogenesis of human cancers. However, the role of BDH2 in gastric cancer (GC) remains largely unclear. Our study aimed to ascertain the regulatory mechanisms of BDH2 in GC, which could be used to develop new therapeutic strategies. METHODS: Western blotting, immunohistochemistry, and RT-PCR were used to investigate the expression of BDH2 in GC specimens and cell lines. Its correlation with the clinicopathological characteristics and prognosis of GC patients was analysed. Functional assays, such as CCK-8 and TUNEL assays, transmission electron microscopy, and an in vivo tumour growth assay, were performed to examine the proliferation, apoptosis, and autophagy of GC cells. Related molecular mechanisms were clarified by luciferase reporter, coimmunoprecipitation, and ubiquitination assays. RESULTS: BDH2 was markedly downregulated in GC tissues and cells, and the low expression of BDH2 was associated with poor survival of GC patients. Functionally, BDH2 overexpression significantly induced apoptosis and autophagy in vitro and in vivo. Mechanistically, BDH2 promoted Keap1 interaction with Nrf2 to increase the ubiquitination level of Nrf2. Ubiquitination/degradation of Nrf2 inhibited the activity of ARE to increase accumulation of reactive oxygen species (ROS), thereby inhibiting the phosphorylation levels of AktSer473 and mTORSer2448. CONCLUSIONS: Our study indicates that BDH2 is an important tumour suppressor in GC. BDH2 regulates intracellular ROS levels to mediate the PI3K/Akt/mTOR pathway through Keap1/Nrf2/ARE signalling, thereby inhibiting the growth of GC.

Participation of Free-Radical Processes in Structural and Metabolic Disturbances in the Lung Tissues Caused by Exposure to Coal-Rock Dust and their Adaptogenic Correction.

Adaptive correction of structural and metabolic disturbances in the lungs caused by longterm exposure to coal-rock dust were studied in experiments on rats. It was shown that the complex antioxidant preparation containing dihydroquercetin compensated disturbances in the redox balance in the lung tissue, prevented the formation of dust granulomas, and reduced the severity of degenerative changes in the bronchopulmonary system.

Inactivation of 3-hydroxybutyrate dehydrogenase type 2 promotes proliferation and metastasis of nasopharyngeal carcinoma by iron retention.

BACKGROUND: 3-Hydroxybutyrate dehydrogenase type 2 (BDH2) is known to catalyse a rate-limiting step in the biogenesis of the mammalian siderophore and regulate intracellular iron metabolism. Here we aim to explore the expression and possible function of BDH2 in nasopharyngeal carcinoma (NPC). METHODS: The transcription and protein expression of BDH2 in NPC were determined by both real-time RT-PCR and immunohistochemistry staining assays. Cell proliferation, migration and invasion were evaluated by MTT assay, wound-healing assay and Transwell assay, respectively. The profile of genes regulated by restoring BDH2 expression in NPC cells was analysed by cDNA microarray. The level of iron in NPC cells was detected by iron colorimetric assay. RESULTS: The expression of BDH2 was significantly downregulated in NPC. Ectopic expression of BDH2 inhibited NPC cell proliferation and colony formation. Meanwhile, BDH2 suppressed the migration and invasion of NPC cells by reversing the epithelial-mesenchymal transition (EMT). In addition, a higher level of BDH2 decreased the growth and metastasis of NPC cells via reducing intracellular iron level. CONCLUSIONS: Our findings suggest that BDH2 may be a candidate tumour-suppressor gene in NPC. Decreasing intracellular iron could be an effective therapeutic approach for NPC.

LncRNA TP73-AS1 sponges miR-141-3p to promote the migration and invasion of pancreatic cancer cells through the up-regulation of BDH2.

LncRNA TP73 antisense RNA 1T (TP73-AS1) plays an important role in human malignancies. However, the levels of TP73-AS1 and its functional mechanisms in pancreatic cancer metastasis remain unknown, and the clinical significance of TP73-AS1 in human pancreatic cancer is also unclear. In the present study, the levels of TP73-AS1 and its candidate target miR-141 in pancreatic cancer and adjacent normal tissue were detected using qRT-PCR. The association between TP73-AS1 levels and the clinicopathologic characteristics of pancreatic cancer patients were analyzed. The relationship between TP73-AS1 and miR-141, and miR-141 and its candidate target 3-hydroxybutyrate dehydrogenase type 2 (BDH2) was confirmed using dual-luciferase reporter assays. TP73-AS1 and/or miR-141 were knocked down using siRNA or an inhibitor in pancreatic cancer cells and cell migration and invasion then examined. The results showed that TP73-AS1 was up-regulated in pancreatic cancer tissue and cell lines. High levels of TP73-AS1 were correlated with poor clinicopathological characteristics and shorter overall survival. MiR-141 was a direct target for TP73-AS1, while BDH2 was a direct target for miR-141. The knockdown of TP73-AS1 significantly inhibited the migration and invasion of pancreatic cancer cells, while the miR-141 inhibitor significantly restored the migration and invasion. Therefore, TP73-AS1 positively regulated BDH2 expression by sponging miR-141. These findings suggest that TP73-AS1 serves as an oncogene and promotes the metastasis of pancreatic cancer. Moreover, TP73-AS1 could serve as a predictor and a potential drug biotarget for pancreatic cancer.

Butyrate regulates inflammatory cytokine expression without affecting oxidative respiration in primary astrocytes from spontaneously hypertensive rats.

Neurons and glia exhibit metabolic imbalances in hypertensive animal models, and loss of metabolic homeostasis can lead to neuroinflammation and oxidative stress. The objective of this study was to determine the effects of the microbial metabolite butyrate on mitochondrial bioenergetics and inflammatory markers in mixed brainstem and hypothalamic primary cultures of astrocytes between normotensive (Sprague-Dawley, S-D) and spontaneously hypertensive (SHR) rats. Bioenergetics of mitochondria in astrocytes from normotensive S-D rats were modified with butyrate, but this was not the case in astrocytes derived from SHR, suggesting aberrant mitochondrial function. Transcripts related to oxidative stress, butyrate transporters, butyrate metabolism, and neuroinflammation were quantified in astrocyte cultures treated with butyrate at 0, 200, 600, and 1000 µmol/L. Butyrate decreased catalase and monocarboxylate transporter 1 mRNA in astrocytes of S-D rats but not in the SHR. Moreover, while butyrate did not directly regulate the expression of 3-hydroxybutyrate dehydrogenase 1 and 2 in astrocytes of either strain, the expression levels for these transcripts in untreated cultures were lower in the SHR compared to S-D. We observed higher levels of specific inflammatory cytokines in astrocytes of SHR, and treatment with butyrate decreased expression of Ccl2 and Tlr4 in SHR astrocytes only. Conversely, butyrate treatment increased expression of tumor necrosis factor in astrocytes from SHR but not from the S-D rats. This study improves our understanding of the role of microbial metabolites in regulating astrocyte function, and provides support that butyrate differentially regulates both the bioenergetics and transcripts related to neuroinflammation in astrocytes from SHR versus S-D rats.

Low ketolytic enzyme levels in tumors predict ketogenic diet responses in cancer cell lines in vitro and in vivo.

The ketogenic diet (KD) is a high-fat, very-low-carbohydrate diet that triggers a fasting state by decreasing glucose and increasing ketone bodies, such as ß-hydroxybutyrate (ßHB). In experimental models and clinical trials, the KD has shown anti-tumor effects, possibly by reducing energy supplies to cells, which damage the tumor microenvironment and inhibit tumor growth. Here, we determined expression levels of genes encoding the ketolytic enzymes 3-hydroxybutyrate dehydrogenase 1 (BDH1) and succinyl-CoA: 3-oxoacid CoA transferase 1 (OXCT1) in 33 human cancer cell lines. We then selected two representative lines, HeLa and PANC-1, for in vivo examination of KD sensitivity in tumors with high or low expression, respectively, of these two enzymes. In mice with HeLa xenografts, the KD increased tumor growth and mouse survival decreased, possibly because these tumors actively consumed ketone bodies as an energy source. Conversely, the KD significantly inhibited growth of PANC-1 xenograft tumors. ßHB added to each cell culture significantly increased proliferation of HeLa cells, but not PANCI-1 cells. Downregulation of both BDH1 and OXCT1 rendered HeLa cells sensitive to the KD in vitro and in vivo. Tumors with low ketolytic enzyme expression may be unable to metabolize ketone bodies, thus predicting a better response to KD therapy.

Downregulation of BDH2 modulates iron homeostasis and promotes DNA demethylation in CD4+ T cells of systemic lupus erythematosus.

DNA hypomethylation plays an important role in the pathogenesis of systemic lupus erythematosus (SLE). Here we investigated whether 3-hydroxy butyrate dehydrogenase 2 (BDH2), a modulator of intracellular iron homeostasis, was involved in regulating DNA hypomethylation and hyper-hydroxymethylation in lupus CD4+ T cells. Our results showed that BDH2 expression was decreased, intracellular iron was increased, global DNA hydroxymethylation level was elevated, while methylation level was reduced in lupus CD4+ T cells compared with healthy controls. The decreased BDH2 contributed to DNA hyper-hydroxymethylation and hypomethylation via increasing intracellular iron in CD4+ T cells, which led to overexpression of immune related genes. Moreover, we showed that BDH2 was the target gene of miR-21. miR-21 promoted DNA demethylation in CD4+ T cells through inhibiting BDH2 expression. Our data demonstrated that the dysregulation of iron homeostasis in CD4+ T cells induced by BDH2 deficiency contributes to DNA demethylation and self-reactive T cells in SLE.

Knockdown of long non-coding RNA TP73-AS1 inhibits cell proliferation and induces apoptosis in esophageal squamous cell carcinoma.

Recent studies have shown that long non-coding RNAs (lncRNAs) are involved in a variety of biological processes and diseases in humans, including cancer. Our study serves as the first comprehensive analysis of lncRNA TP73-AS1 in esophageal cancer. We utilized a lncRNA microarray to analyze the expression profile of lncRNAs in esophageal squamous cell carcinoma. Our results show that lncRNA TP73-AS1 and BDH2 levels are generally upregulated in esophageal cancer tissues and are strongly correlated with tumor location or TNM stage in clinical samples. LncRNA TP73-AS1 knockdown inhibited BDH2 expression in EC9706 and KYSE30 cells, whereas BDH2 knockdown repressed esophageal cancer cell proliferation and induced apoptosis via the caspase-3 dependent apoptotic pathway. Overexpression of BDH2 in lncRNA TP73-AS1 knockdown cells partially rescued cell proliferation rates and suppressed apoptosis. In mouse xenografts, tumor size was reduced in lncRNA TP73-ASI siRNA-transfected tumors, suggesting that downregulation of lncRNA TP73-AS1 attenuated EC proliferation in vitro and in vivo. In addition, BDH2 or lncRNA TP73-AS1 knockdown enhanced the chemosensitivity of esophageal cancer cells to 5-FU and cisplatin. Our results suggest that lncRNA TP73-AS1 may be a novel prognostic biomarker that could serve as a potential therapeutic target for the treatment of esophageal cancer.

Regulation of mammalian siderophore 2,5-DHBA in the innate immune response to infection.

Competition for iron influences host-pathogen interactions. Pathogens secrete small iron-binding moieties, siderophores, to acquire host iron. In response, the host secretes siderophore-binding proteins, such as lipocalin 24p3, which limit siderophore-mediated iron import into bacteria. Mammals produce 2,5-dihydroxy benzoic acid, a compound that resembles a bacterial siderophore. Our data suggest that bacteria use both mammalian and bacterial siderophores. In support of this idea, supplementation with mammalian siderophore enhances bacterial growth in vitro. In addition, mice lacking the mammalian siderophore resist E. coli infection. Finally, we show that the host responds to infection by suppressing siderophore synthesis while up-regulating lipocalin 24p3 expression via TLR signaling. Thus, reciprocal regulation of 24p3 and mammalian siderophore is a protective mechanism limiting microbial access to iron.

3-Hydroxybutyrate dehydrogenase-2 and ferritin-H synergistically regulate intracellular iron.

Siderophores are best known as small iron-binding molecules that facilitate iron uptake in bacteria and fungi. In our previous study, we demonstrated that eukaryotes also produce siderophore-like molecules via a remarkably conserved biosynthetic pathway. A member of the short-chain dehydrogenase family of reductases, 3-hydroxybutyrate dehydrogenase-2, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore. Physiologically, depletion of the mammalian siderophore by inhibiting expression of the 3-hydroxybutyrate dehydrogenase-2 gene (Bdh2) results in abnormal accumulation of intracellular iron, increased oxidative stress, and mitochondrial iron deficiency. Thus, the mammalian siderophore is an important regulator of cellular iron homeostasis. The cellular iron storage protein ferritin also regulates iron metabolism and protects cells from oxidative stress. Depletion of ferritin results in intracellular iron accumulation, predisposes to oxidative stress, and confers a growth advantage to cells. We therefore hypothesize that the siderophore and ferritin coregulate cellular iron metabolism/homeostasis in eukaryotes. We tested this prediction by depleting both the siderophore and ferritin. This resulted in a marked accumulation of cellular iron, and caused increased sensitivity to oxidants. Interestingly, cells lacking both the siderophore and ferritin proliferated at a higher rate than cells lacking either of these components alone. Taken together, our findings suggest that the siderophore and ferritin synergistically regulate cellular iron levels.

Inflammation and ER stress downregulate BDH2 expression and dysregulate intracellular iron in macrophages.

Macrophages play a very important role in host defense and in iron homeostasis by engulfing senescent red blood cells and recycling iron. Hepcidin is the master iron regulating hormone that limits dietary iron absorption from the gut and limits iron egress from macrophages. Upon infection macrophages retain iron to limit its bioavailability which limits bacterial growth. Recently, a short chain butyrate dehydrogenase type 2 (BDH2) protein was reported to contain an iron responsive element and to mediate cellular iron trafficking by catalyzing the synthesis of the mammalian siderophore that binds labile iron; therefore, BDH2 plays a crucial role in intracellular iron homeostasis. However, BDH2 expression and regulation in macrophages have not yet been described. Here we show that LPS-induced inflammation combined with ER stress led to massive BDH2 downregulation, increased the expression of ER stress markers, upregulated hepcidin expression, downregulated ferroportin expression, caused iron retention in macrophages, and dysregulated cytokine release from macrophages. We also show that ER stress combined with inflammation synergistically upregulated the expression of the iron carrier protein NGAL and the stress-inducible heme degrading enzyme heme oxygenase-1 (HO-1) leading to iron liberation. This is the first report to show that inflammation and ER stress downregulate the expression of BDH2 in human THP-1 macrophages.

Human BDH2, an anti-apoptosis factor, is a novel poor prognostic factor for de novo cytogenetically normal acute myeloid leukemia.

BACKGROUND: The relevance of recurrent molecular abnormalities in cytogenetically normal (CN) acute myeloid leukemia (AML) was recently acknowledged by the inclusion of molecular markers such as NPM1, FLT3, and CEBPA as a complement to cytogenetic information within both the World Health Organization and the European Leukemia Net classifications. Mitochondrial metabolism is different in cancer and normal cells. A novel cytosolic type 2-hydroxybutyrate dehydrogenase, BDH2, originally named DHRS6, plays a physiological role in the cytosolic utilization of ketone bodies, which can subsequently enter mitochondria and the tricarboxylic acid cycle. Moreover, BDH2 catalyzes the production of 2, 3-DHBA during enterobactin biosynthesis and participates in 24p3 (LCN2)-mediated iron transport and apoptosis. RESULTS: We observed that BDH2 expression is an independent poor prognostic factor for CN-AML, with an anti-apoptotic role. Patients with high BDH2 expression have relatively shorter overall survival (P = 0.007) and a low complete response rate (P = 0.032). BDH2-knockdown (BDH2-KD) in THP1 and HL60 cells increased the apoptosis rate under reactive oxygen species stimulation. Decrease inducible survivin, a member of the inhibitors of apoptosis family, but not members of the Bcl-2 family, induced apoptosis via a caspase-3-independent pathway upon BDH2-KD. CONCLUSIONS: BDH2 is a novel independent poor prognostic marker for CN-AML, with the role of anti-apoptosis, through surviving.