A Small-Molecule Inhibitor of Factor Inhibiting HIF Binding to a Tyrosine-flip Pocket for the Treatment of Obesity.

In animals limiting oxygen upregulates hypoxia-inducible factor (HIF) promoting a metabolic shift towards glycolysis. Factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that regulates HIF function by reducing its interaction with histone acetyl transferases. HIF levels are negatively regulated by the HIF prolyl hydroxylases (PHDs), which like FIH, are 2-oxoglutarate(2OG) oxygenases. Genetic loss of FIH promotes both glycolysis and aerobic metabolism. FIH has multiple non-HIF substrates making it challenging to connect its biochemistry with physiology. A structure-mechanism guided approach identified a highly potent in vivo active FIH inhibitor, ZG-2291, binding of which promotes a conformational flip of a catalytically important tyrosine, enabling selective inhibition of FIH over other JmjC subfamily 2OG oxygenases. Consistent with genetic studies, ZG-2291 promotes thermogenesis and ameliorates symptoms of obesity and metabolic dysfunction in ob/ob mice. The results reveal ZG-2291 as a useful probe for the physiological functions of FIH and identify FIH inhibition as a promising strategy for obesity treatment.

Biochemical and Structural Insights into FIH-Catalysed Hydroxylation of Transient Receptor Potential Ankyrin Repeat Domains.

Transient receptor potential (TRP) channels have important roles in environmental sensing in animals. Human TRP subfamily A member 1 (TRPA1) is responsible for sensing allyl isothiocyanate (AITC) and other electrophilic sensory irritants. TRP subfamily vanilloid member 3 (TRPV3) is involved in skin maintenance. TRPV3 is a reported substrate of the 2-oxoglutarate oxygenase factor inhibiting hypoxia-inducible factor (FIH). We report biochemical and structural studies concerning asparaginyl hydroxylation of the ankyrin repeat domains (ARDs) of TRPA1 and TRPV3 catalysed by FIH. The results with ARD peptides support a previous report on FIH-catalysed TRPV3 hydroxylation and show that, of the 12 potential TRPA1 sequences investigated, one sequence (TRPA1 residues 322-348) undergoes hydroxylation at Asn336. Structural studies reveal that the TRPA1 and TRPV3 ARDs bind to FIH with a similar overall geometry to most other reported FIH substrates. However, the binding mode of TRPV3 to FIH is distinct from that of other substrates.

Evolution of hypoxia and hypoxia-inducible factor asparaginyl hydroxylase regulation in chronic kidney disease.

BACKGROUND: The roles of hypoxia and hypoxia inducible factor (HIF) during chronic kidney disease (CKD) are much debated. Interventional studies with HIF-α activation in rodents have yielded contradictory results. The HIF pathway is regulated by prolyl and asparaginyl hydroxylases. While prolyl hydroxylase inhibition is a well-known method to stabilize HIF-α, little is known about the effect asparaginyl hydroxylase factor inhibiting HIF (FIH). METHODS: We used a model of progressive proteinuric CKD and a model of obstructive nephropathy with unilateral fibrosis. In these models we assessed hypoxia with pimonidazole and vascularization with three-dimensional micro-computed tomography imaging. We analysed a database of 217 CKD biopsies from stage 1 to 5 and we randomly collected 15 CKD biopsies of various severity degrees to assess FIH expression. Finally, we modulated FIH activity in vitro and in vivo using a pharmacologic approach to assess its relevance in CKD. RESULTS: In our model of proteinuric CKD, we show that early CKD stages are not characterized by hypoxia or HIF activation. At late CKD stages, some areas of hypoxia are observed, but these are not colocalizing with fibrosis. In mice and in humans, we observed a downregulation of the HIF pathway, together with an increased FIH expression in CKD, according to its severity. Modulating FIH in vitro affects cellular metabolism, as described previously. In vivo, pharmacologic FIH inhibition increases the glomerular filtration rate of control and CKD animals and is associated with decreased development of fibrosis. CONCLUSIONS: The causative role of hypoxia and HIF activation in CKD progression is questioned. A pharmacological approach of FIH downregulation seems promising in proteinuric kidney disease.

High-altitude deer mouse hypoxia-inducible factor-2α shows defective interaction with CREB-binding protein.

Numerous mammalian species have adapted to the chronic hypoxia of high altitude. Recent genomic studies have identified evidence for natural selection of genes and associated genetic changes in these species. A major gap in our knowledge is an understanding of the functional significance, if any, of these changes. Deer mice (Peromyscus maniculatus) live at both low and high altitudes in North America, providing an opportunity to identify functionally important genetic changes. High-altitude deer mice show evidence of natural selection on the Epas1 gene, which encodes for hypoxia-inducible factor-2α (Hif-2α), a central transcription factor of the hypoxia-inducible factor pathway. An SNP encoding for a T755M change in the Hif-2α protein is highly enriched in high-altitude deer mice, but its functional significance is unknown. Here, using coimmunoprecipitation and transcriptional activity assays, we show that the T755M mutation produces a defect in the interaction of Hif-2α with the transcriptional coactivator CREB-binding protein. This results in a loss of function because of decreased transcriptional activity. Intriguingly, the effect of this mutation depends on the amino acid context. Interchanges between methionine and threonine at the corresponding position in house mouse (Mus musculus) Hif-2α are without effects on CREB-binding protein binding. Furthermore, transfer of a set of deer mouse-specific Hif-2α amino acids to house mouse Hif-2α is sufficient to confer sensitivity of house mouse Hif-2α to the T755M substitution. These findings provide insight into high-altitude adaptation in deer mice and evolution at the Epas1 locus.

Structural and thermodynamical insights into the binding and inhibition of FIH-1 by the N-terminal disordered region of Mint3.

Mint3 is known to enhance aerobic ATP production, known as the Warburg effect, by binding to FIH-1. Since this effect is considered to be beneficial for cancer cells, the interaction is a promising target for cancer therapy. However, previous research has suggested that the interacting region of Mint3 with FIH-1 is intrinsically disordered, which makes investigation of this interaction challenging. Therefore, we adopted thermodynamic and structural studies in solution to clarify the structural and thermodynamical changes of Mint3 binding to FIH-1. First, using a combination of circular dichroism, nuclear magnetic resonance, and hydrogen/deuterium exchange-mass spectrometry (HDX-MS), we confirmed that the N-terminal half, which is the interacting part of Mint3, is mostly disordered. Next, we revealed a large enthalpy and entropy change in the interaction of Mint3 using isothermal titration calorimetry (ITC). The profile is consistent with the model that the flexibility of disordered Mint3 is drastically reduced upon binding to FIH-1. Moreover, we performed a series of ITC experiments with several types of truncated Mint3s, an effective approach since the interacting part of Mint3 is disordered, and identified amino acids 78 to 88 as a novel core site for binding to FIH-1. The truncation study of Mint3 also revealed the thermodynamic contribution of each part of Mint3 to the interaction with FIH-1, where the core sites contribute to the affinity (ΔG), while other sites only affect enthalpy (ΔH), by forming noncovalent bonds. This insight can serve as a foothold for further investigation of intrinsically disordered regions (IDRs) and drug development for cancer therapy.

OTUB1 regulates lung development, adult lung tissue homeostasis, and respiratory control.

OTUB1 is one of the most highly expressed deubiquitinases, counter-regulating the two most abundant ubiquitin chain types. OTUB1 expression is linked to the development and progression of lung cancer and idiopathic pulmonary fibrosis in humans. However, the physiological function of OTUB1 is unknown. Here, we show that constitutive whole-body Otub1 deletion in mice leads to perinatal lethality by asphyxiation. Analysis of (single-cell) RNA sequencing and proteome data demonstrated that OTUB1 is expressed in all lung cell types with a particularly high expression during late-stage lung development (E16.5, E18.5). At E18.5, the lungs of animals with Otub1 deletion presented with increased cell proliferation that decreased saccular air space and prevented inhalation. Flow cytometry-based analysis of E18.5 lung tissue revealed that Otub1 deletion increased proliferation of major lung parenchymal and mesenchymal/other non-hematopoietic cell types. Adult mice with conditional whole-body Otub1 deletion (wbOtub1del/del ) also displayed increased lung cell proliferation in addition to hyperventilation and failure to adapt the respiratory pattern to hypoxia. On the molecular level, Otub1 deletion enhanced mTOR signaling in embryonic and adult lung tissues. Based on these results, we propose that OTUB1 is a negative regulator of mTOR signaling with essential functions for lung cell proliferation, lung development, adult lung tissue homeostasis, and respiratory regulation.

MicroRNA-122-5p ameliorates tubular injury in diabetic nephropathy via FIH-1/HIF-1α pathway.

Diabetes kidney disease (DKD) affects approximately one-third of diabetes patients, however, the specific molecular mechanism of DKD remains unclear, and there is still a lack of effective therapies. Here, we demonstrated a significant increase of microRNA-122-5p (miR-122-5p) in renal tubular cells in STZ induced diabetic nephropathy (DN) mice. Moreover, inhibition of miR-122-5p led to increased cell death and serve tubular injury and promoted DN progression following STZ treatment in mice, whereas supplementation of miR-122-5p mimic had kidney protective effects in this model. In addition, miR-122-5p suppressed the expression of factor inhibiting hypoxia-inducible factor-1 (FIH-1) in vitro models of DN. microRNA target reporter assay further verified FIH-1 as a direct target of miR-122-5p. Generally, FIH-1 inhibits the activity of HIF-1α. Our in vitro study further indicated that overexpression of HIF-1α by transfection of HIF-1α plasmid reduced tubular cell death, suggesting a protective role of HIF-1α in DN. Collectively, these findings may unveil a novel miR-122-5p/FIH-1/HIF-1α pathway which can attenuate the DN progression.

The Deubiquitinase OTUB1 Is a Key Regulator of Energy Metabolism.

Dysregulated energy metabolism is a major contributor to a multitude of pathologies, including obesity and diabetes. Understanding the regulation of metabolic homeostasis is of utmost importance for the identification of therapeutic targets for the treatment of metabolically driven diseases. We previously identified the deubiquitinase OTUB1 as substrate for the cellular oxygen sensor factor-inhibiting HIF (FIH) with regulatory effects on cellular energy metabolism, but the physiological relevance of OTUB1 is unclear. Here, we report that the induced global deletion of OTUB1 in adult mice (Otub1 iKO) elevated energy expenditure, reduced age-dependent body weight gain, facilitated blood glucose clearance and lowered basal plasma insulin levels. The respiratory exchange ratio was maintained, indicating an unaltered nutrient oxidation. In addition, Otub1 deletion in cells enhanced AKT activity, leading to a larger cell size, higher ATP levels and reduced AMPK phosphorylation. AKT is an integral part of insulin-mediated signaling and Otub1 iKO mice presented with increased AKT phosphorylation following acute insulin administration combined with insulin hypersensitivity. We conclude that OTUB1 is an important regulator of metabolic homeostasis.

Oxygen-dependent asparagine hydroxylation of the ubiquitin-associated (UBA) domain in Cezanne regulates ubiquitin binding.

Deubiquitinases (DUBs) are vital for the regulation of ubiquitin signals, and both catalytic activity of and target recruitment by DUBs need to be tightly controlled. Here, we identify asparagine hydroxylation as a novel posttranslational modification involved in the regulation of Cezanne (also known as OTU domain-containing protein 7B (OTUD7B)), a DUB that controls key cellular functions and signaling pathways. We demonstrate that Cezanne is a substrate for factor inhibiting HIF1 (FIH1)- and oxygen-dependent asparagine hydroxylation. We found that FIH1 modifies Asn35 within the uncharacterized N-terminal ubiquitin-associated (UBA)-like domain of Cezanne (UBACez), which lacks conserved UBA domain properties. We show that UBACez binds Lys11-, Lys48-, Lys63-, and Met1-linked ubiquitin chains in vitro, establishing UBACez as a functional ubiquitin-binding domain. Our findings also reveal that the interaction of UBACez with ubiquitin is mediated via a noncanonical surface and that hydroxylation of Asn35 inhibits ubiquitin binding. Recently, it has been suggested that Cezanne recruitment to specific target proteins depends on UBACez Our results indicate that UBACez can indeed fulfill this role as regulatory domain by binding various ubiquitin chain types. They also uncover that this interaction with ubiquitin, and thus with modified substrates, can be modulated by oxygen-dependent asparagine hydroxylation, suggesting that Cezanne is regulated by oxygen levels.

FIH-1-modulated HIF-1α C-TAD promotes acute kidney injury to chronic kidney disease progression via regulating KLF5 signaling.

Incomplete recovery from episodes of acute kidney injury (AKI) can predispose patients to develop chronic kidney disease (CKD). Although hypoxia-inducible factor-1α (HIF-1α) is a master regulator of the response to hypoxia/ischemia, the role of HIF-1α in CKD progression following incomplete recovery from AKI is poorly understood. Here, we investigated this issue using moderate and severe ischemia/reperfusion injury (I/RI) mouse models. We found that the outcomes of AKI were highly associated with the time course of tubular HIF-1α expression. Sustained activation of HIF-1α, accompanied by the development of renal fibrotic lesions, was found in kidneys with severe AKI. The AKI to CKD progression was markedly ameliorated when PX-478 (a specific HIF-1α inhibitor, 5 mg· kg-1·d-1, i.p.) was administered starting on day 5 after severe I/RI for 10 consecutive days. Furthermore, we demonstrated that HIF-1α C-terminal transcriptional activation domain (C-TAD) transcriptionally stimulated KLF5, which promoted progression of CKD following severe AKI. The effect of HIF-1α C-TAD activation on promoting AKI to CKD progression was also confirmed in in vivo and in vitro studies. Moreover, we revealed that activation of HIF-1α C-TAD resulted in the loss of FIH-1, which was the key factor governing HIF-1α-driven AKI to CKD progression. Overexpression of FIH-1 inhibited HIF-1α C-TAD and prevented AKI to CKD progression. Thus, FIH-1-modulated HIF-1α C-TAD activation was the key mechanism of AKI to CKD progression by transcriptionally regulating KLF5 pathway. Our results provide new insights into the role of HIF-1α in AKI to CKD progression and also the potential therapeutic strategy for the prevention of renal diseases progression.

miR-125 family regulates XIRP1 and FIH in response to myocardial infarction.

MicroRNAs (miRNAs) are powerful regulators of protein expression. Many play important roles in cardiac development and disease. While several miRNAs and targets have been well characterized, the abundance of miRNAs and the numerous potential targets for each suggest that the vast majority of these interactions have yet to be described. The goal of this study was to characterize miRNA expression in the mouse heart after coronary artery ligation (LIG) and identify novel mRNA targets altered during the initial response to ischemic stress. We performed small RNA sequencing (RNA-Seq) of ischemic heart tissue 1 day and 3 days after ligation and identified 182 differentially expressed miRNAs. We then selected relevant mRNA targets from all potential targets by correlating miRNA and mRNA expression from a corresponding RNA-Seq data set. From this analysis we chose to focus, as proof of principle, on two miRNAs from the miR-125 family, miR-125a and miR-351, and two of their potential mRNA targets, Xin actin-binding repeat-containing protein 1 (XIRP1) and factor inhibiting hypoxia-inducible factor (FIH). We found miR-125a to be less abundant and XIRP1 more abundant after ligation. In contrast, the related murine miRNA miR-351 was substantially upregulated in response to ischemic injury, and FIH expression correspondingly decreased. Luciferase reporter assays confirmed direct interactions between these miRNAs and targets. In summary, we utilized a correlative analysis strategy combining miRNA and mRNA expression data to identify functional miRNA-mRNA relationships in the heart after ligation. These findings provide insight into the response to ischemic injury and suggest future therapeutic targets.

Nuclear entry and export of FIH are mediated by HIF1α and exportin1, respectively.

Hypoxia plays a crucial role at cellular and physiological levels in all animals. The responses to chronic hypoxia are, at least substantially, orchestrated by activation of the hypoxia inducible transcription factors (HIFs), whose stability and subsequent transcriptional activation are regulated by HIF hydroxylases. Factor inhibiting HIF (FIH), initially isolated as a HIFα interacting protein following a yeast two-hybrid screen, is an asparaginyl hydroxylase that negatively regulates transcriptional activation by HIF. This study aimed to define the mechanisms that govern transitions of FIH between the nucleus and cytoplasm. We report that FIH accumulates in the nucleus within a short time window during hypoxia treatment. We provide evidence, based on the application of genetic interventions and small molecule inhibition of the HIF hydroxylases, that the nuclear localization of FIH is governed by two opposing processes: nuclear entry by 'coupling' with HIF1α for importin ß1-mediated nuclear import and active export via a Leptomycin B-sensitive exportin1-dependent pathway.This article has an associated First Person interview with the first author of the paper.

Role of Microtubule-Associated Factors in HIF1α Nuclear Translocation.

Adaptation to hypoxia is essential for regulating the survival and functions of hypoxic cells; it is mainly mediated by the hypoxia-inducible factor 1 (HIF1). The alpha subunit of HIF1 (HIF1α) is a well-known regulatory component of HIF1, which is tightly controlled by various types of HIF1α-regulating processes. Previous research has shown that microtubule-regulated HIF1α nuclear translocation is a key factor for HIF1 activation under hypoxia. In this review, we summarize experimental reports on the role of microtubule-associated factors, such as microtubule, dynein, and dynein adaptor protein, in nuclear translocation of HIF1α. Based upon scientific evidence, we propose a bicaudal D homolog (BICD) as a novel HIF1α translocation regulating factor. A deeper understanding of the mechanism of the action of regulatory factors in controlling HIF1α nuclear translocation will provide novel insights into cell biology under hypoxia.

Activation of AMPK under Hypoxia: Many Roads Leading to Rome.

AMP-activated protein kinase (AMPK) is known as a pivotal cellular energy sensor, mediating the adaptation to low energy levels by deactivating anabolic processes and activating catabolic processes in order to restore the cellular ATP supply when the cellular AMP/ATP ratio is increased. Besides this well-known role, it has also been shown to exert protective effects under hypoxia. While an insufficient supply with oxygen might easily deplete cellular energy levels, i.e., ATP concentration, manifold other mechanisms have been suggested and are heavily disputed regarding the activation of AMPK under hypoxia independently from cellular AMP concentrations. However, an activation of AMPK preceding energy depletion could induce a timely adaptation reaction preventing more serious damage. A connection between AMPK and the master regulator of hypoxic adaptation via gene transcription, hypoxia-inducible factor (HIF), has also been taken into account, orchestrating their concerted protective action. This review will summarize the current knowledge on mechanisms of AMPK activation under hypoxia and its interrelationship with HIF.

Expression of HIF-2a in clear-cell renal cell carcinoma independently predicts overall survival.

The purpose of this study is to evaluate the expression and the prognostic role of main factors, involved in the hypoxia pathway, in patients with clear-cell renal cell carcinoma (ccRCC). Immunohistochemical expression of Hypoxia inducible factors (HIF) HIF-1a, HIF-2a, prolyl hydroxylases PHD1, PHD2, PHD3, and factor inhibiting HIF (FIH) was assessed on a tissue microarray, containing tumour and corresponding normal kidney tissue from 66 patients underwent surgery for ccRCC. Expression levels were evaluated in relation to T stage, Fuhrman grade, cancer-specific, and overall survival (OS). Cytoplasmatic expression of HIF-2a was positively correlated with expression of HIF-1a (p = 0.011). HIF-1a expression was also positively correlated with PHD3 and FIH (p = 0.020 and p = 0.039). Expression of HIF-1a was associated with lower Fuhrman grade (p = 0.008), while HIF-2a overexpression with unfavourable grade (p = 0.026). PHD3 was significant downregulated (84.8%). Age, LDH, presence of necrosis, Fuhrman grade, T stage, and HIF-2a cytoplasmatic expression were significant associated with OS of patients in univariable analysis. In multivariable analysis, HIF-2a expression (p = 0.006) and T stage (p = 0.001) remained as the only independent predictors for overall survival. These results indicate that HIF-2a overexpression not only is inversely correlated with Fuhrman grade in ccRCC, but also represents a strong independent prognostic factor for a poor overall survival.

Cognitive Enhancer Noopept Activates Transcription Factor HIF-1.

The effect of noopept (N-phenylacetyl-L-prolyl-glycine ethyl ester) on the DNA-binding activity of HIF-1 in SH-SH5Y cells and the mechanisms of stabilization of this transcription factor were studied in vitro. Noopept was shown to increase both the basal DNA-binding activity of HIF-1 and the activity induced by various hypoxia mimetics. The mechanism of stabilization of the oxygen-sensitive HIF1α subunit by noopept involves the inhibition of HIF-1 prolyl hydroxylase, which is indirectly indicated by the data obtained using the ODD-Luc reporter, and the positive effect on the level of the HIF1α protein. It was revealed that the effect of noopept is accompanied by changes in gene expression, which belong to different metabolic pathways and are controlled by the transcription factor HIF-1.

Deletion of the fih gene encoding an inhibitor of hypoxia-inducible factors increases hypoxia tolerance in zebrafish.

Many aerobic organisms have developed molecular mechanism to tolerate hypoxia, but the specifics of these mechanisms remain poorly understood. It is important to develop genetic methods that confer increased hypoxia tolerance to intensively farmed aquatic species, as these are maintained in environments with limited available oxygen. As an asparaginyl hydroxylase of hypoxia-inducible factors (HIFs), factor inhibiting HIF (FIH) inhibits transcriptional activation of hypoxia-inducible genes by blocking the association of HIFs with the transcriptional coactivators CREB-binding protein (CBP) and p300. Therefore, here we sought to test whether fih is involved in regulating hypoxia tolerance in the commonly used zebrafish model. Overexpressing the zebrafish fih gene in epithelioma papulosum cyprini (EPC) cells and embryos, we found that fih inhibits the transcriptional activation of zebrafish HIF-α proteins. Using CRISPR/Cas9 to obtain fih-null zebrafish mutants, we noted that the fih deletion makes zebrafish more tolerant of hypoxic conditions than their WT siblings, but does not result in oxygen consumption rates that significantly differ from those of WT fish. Of note, we identified fewer apoptotic cells in adult fih-null zebrafish brains and in fih-null embryos, possibly explaining why the fih-null mutant had greater hypoxia tolerance than the WT. Moreover, the fih deletion up-regulated several hypoxia-inducible genes in fih-null zebrafish exposed to hypoxia. The findings of our study suggest that fih plays a role in hypoxia tolerance by affecting the rate of cellular apoptosis in zebrafish.

The functional interplay between the HIF pathway and the ubiquitin system - more than a one-way road.

The hypoxia inducible factor (HIF) pathway and the ubiquitin system represent major cellular processes that are involved in the regulation of a plethora of cellular signaling pathways and tissue functions. The ubiquitin system controls the ubiquitination of proteins, which is the covalent linkage of one or several ubiquitin molecules to specific targets. This ubiquitination is catalyzed by approximately 1000 different E3 ubiquitin ligases and can lead to different effects, depending on the type of internal ubiquitin chain linkage. The best-studied function is the targeting of proteins for proteasomal degradation. The activity of E3 ligases is antagonized by proteins called deubiquitinases (or deubiquitinating enzymes), which negatively regulate ubiquitin chains. This is performed in most cases by the catalytic removal of these chains from the targeted protein. The HIF pathway is regulated in an oxygen-dependent manner by oxygen-sensing hydroxylases. Covalent modification of HIFα subunits leads to the recruitment of an E3 ligase complex via the von Hippel-Lindau (VHL) protein and the subsequent polyubiquitination and proteasomal degradation of HIFα subunits, demonstrating the regulation of the HIF pathway by the ubiquitin system. This unidirectional effect of an E3 ligase on the HIF pathway is the best-studied example for the interplay between these two important cellular processes. However, additional regulatory mechanisms of the HIF pathway through the ubiquitin system are emerging and, more recently, also the reciprocal regulation of the ubiquitin system through components of the HIF pathway. Understanding these mechanisms and their relevance for the activity of each other is of major importance for the comprehensive elucidation of the oxygen-dependent regulation of cellular processes. This review describes the current knowledge of the functional bidirectional interplay between the HIF pathway and the ubiquitin system on the protein level.

Study on the role of Hsa-miR-31-5p in hypertrophic scar formation and the mechanism.

Hypertrophic scar (HS) formation is associated with the fibrosis of fibrocytes caused by excessive extracellular matrix (ECM) synthesis and deposition, the initial event of HS formation. Our high throughput screen of miRNA expression profiles identified hsa-miR31-5p, whose transcription level was most differentially in normal skin fibroblasts (NS) and HS among other miRNAs. The level of hsa-miR31-5p in HS was significantly higher than in NS. In-vitro functional experiments showed hsa-miR31-5p knockdown remarkably suppressed the proliferation of hypertrophic scar fibroblasts (HSFBs) under hypoxia, promoted cell invasion, and inhibited the expression of Collagen I and III and Fibronectin (FN), suggesting that hsa-miR31-5p knockdown effectively reduces HS formation caused by excessive ECM synthesis and deposition in HSFBs under hypoxia. Mechanism study showed that the regulation of HS formation by hsa-miR31-5p was mediated by its target gene, factor-inhibiting HIF-1 (FIH): under hypoxia, hsa-miR31-5p down-regulated FIH and thus increased the level of hypoxia inducible factor-1α (HIF-1α), which subsequently activated the HIF-1α fibrosis regulation pathway in HSFBs, and stimulated the proliferation and ECM synthesis in HSFBs, eventually resulting in fibrosis and scar formation. The data also show that knockdown of hsa-miR31-5p in HSFBs impaired the trend of increased proliferation, reduced invasion and excessive ECM synthesis and deposition caused by HIF-1a activation under hypoxia through upregulating FIH, indicating that knockdown of hsa-miR31-5p effectively inhibits the formation of HS. In conclusion, hsa-miR31 -5p plays an important role in HS formation by inhibiting FIH and regulating the HIF-1α pathway. Therefore, hsa-miR31 -5p may be a novel therapeutic target for HS.

Radioprotective Activity and Preliminary Mechanisms of N-oxalyl-d-phenylalanine (NOFD) In Vitro.

The radiation-induced damage to the human body is primarily caused by excessive reactive oxygen species (ROS) production after irradiation. Therefore, the removal of the increase of ROS caused by ionizing radiation (IR) has been the focus of research on radiation damage protective agents. Hypoxia inducible factor (HIF) is a transcription factor in human and plays an important role in regulating the body metabolism. Factor inhibiting HIF (FIH) is an endogenous inhibitor factor of HIF protein under normoxia conditions. It has been shown that the high expression of HIF protein has a certain repair effect on radiation-induced intestinal injury and hematopoietic system damage in mice; however, it is not clear about the effect of HIF on the level of ROS after radiation. In this study, the role of N-oxalyl-d-phenylalanine (NOFD), an FIH inhibitor, for its effect on alleviating ROS level is investigated in the cells. Our results indicate that pretreatment with NOFD can mitigate ROS level and alleviate IR-induced DNA damage and apoptosis in vitro. Therefore, HIF can be used as a target on scavengers. Furthermore, in order to explore the relevant mechanism, we also test the expression of relevant HIF downstream genes in the cells, finding that Notch-2 gene is more sensitive to NOFD treatment. This experiment result is used to support the subsequent mechanism experiments.