LEF-1 Regulates C/EBP α Expression and Neutrophil Differentiation in Normal Myelopoiesis and Congenital Neutropenia by a β-Catenin Independent Mechanism.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 500-500 ◽  
Author(s):  
Julia Skokowa ◽  
Murat Uenalan ◽  
Axel Schambach ◽  
Michaela Scherr ◽  
Matthias Eder ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Molecular Mechanisms ◽  
Wnt Signaling Pathway ◽  
Binding Domain ◽  
Granulocytic Differentiation ◽  
Congenital Neutropenia ◽  
Cd34 Cells ◽  
Binding Factor ◽  
Myeloid Progenitors

Abstract Lymphoid enhancer-binding factor 1 (LEF-1) belongs to the canonical Wnt signaling pathway acting in transcriptional complexes with β-catenin. LEF-1 can also act independent of β-catenin (i.e. in the TGF-β or Notch pathway). Additionally, recent studies described LEF-1 dominant negative isoform (dnLEF-1), which lack the β-catenin binding domain and functions as either a transcriptional repressor or activator. To date, analysis of the role of LEF-1 in hematopoiesis has been restricted to the lymphoid compartment. Previously we described the crucial role of lymphoid enhancer-binding factor 1 (LEF-1) in granulopoiesis. We found that LEF-1 mediates proliferation, survival, and differentiation of granulocyte progenitor cells. Moreover, abrogated LEF-1 expression is one of the pathomechanism of severe congenital neutropenia CN (Skokowa et al., Nature Medicine, in press). Based on these findings, we aimed to characterize the molecular mechanisms of LEF-1 in the regulation of granulocytic differentiation. C/EBPα is well known as a key transcription factor in granulopoiesis and we found it to be a target gene directly regulated by LEF-1. A screen of the known 566 bp upstream promoter of C/EBPα gene revealed a putative LEF-1 binding site (− 559 bp to − 538 bp). We confirmed LEF-1 binding to C/EBPα promoter in nuclear extracts from CD34+ and CD33+ cells in the transcription factor binding NoShift and ChIP assays. Interestingly, LEF-1 binds to the C/EBPα promoter more efficiently in CD33+ myeloid progenitors than in CD34+ cells. The direct regulation of C/EBPα by LEF-1 was further confirmed in functional studies. We found that in line with down-regulation of LEF-1, expression of C/EBPα was also significantly reduced in CD33+ myeloid progenitors of CN patients. Moreover, LEF-1 rescue of these cells resulted in a marked up-regulation of C/EBPα mRNA expression and in vitro restoration of defective granulocytic differentiation. Remarkably, transduction of CN CD33+ cells with dnLEF-1 isoform, which lacks the ß-catenin-binding domain, resulted in up-regulation of C/EBPα to a similar degree as it was observed with full-length LEF-1. A direct regulatory link between LEF-1 and C/EBPα was additionally confirmed in LEF-1 inhibition experiments. C/EBPα expression was significantly down-regulated in CD34+ cells of healthy individuals, transduced with LEF-1 shRNA. Therefore, we propose that LEF-1 is a key regulator of myeloid differentiation acting in a β-catenin-independent manner, similar as it is known for LEF-1 regulation of T-lymphocyte development.

Neutrophil elastase is severely down-regulated in severe congenital neutropenia independent of ELA2 or HAX1 mutations but dependent on LEF-1

Blood ◽  
2009 ◽  
Vol 114 (14) ◽  
pp. 3044-3051 ◽  
Author(s):  
Julia Skokowa ◽  
John Paul Fobiwe ◽  
Lan Dan ◽  
Basant Kumar Thakur ◽  
Karl Welte
Keyword(s):  
Neutrophil Elastase ◽  
Gene Promoter ◽  
Severe Congenital Neutropenia ◽  
Granulocytic Differentiation ◽  
Congenital Neutropenia ◽  
Protein Levels ◽  
Cd34 Cells ◽  
Direct Binding ◽  
Myeloid Progenitors

Abstract Severe congenital neutropenia (CN) is a heterogeneous disorder of myelopoiesis which follows an autosomal dominant or autosomal recessive pattern of inheritance. Genetic analyses indicate mutations in the ELA2 gene in most patients. We have identified LEF-1 as a decisive transcription factor in granulopoiesis controlling proliferation and granulocytic differentiation by direct activation of its target gene, C/EBPα. In patients with CN, the expression of LEF-1 and C/EBPα was abrogated in myeloid progenitors leading to maturation arrest of granulopoiesis. In the present study we demonstrated that ELA2 mRNA expression in myeloid progenitors and plasma protein levels of neutrophil elastase (NE) were markedly reduced in patients with CN harboring mutations in either ELA2 or HAX-1 genes. The ELA2 gene promoter is positively regulated by the direct binding of LEF-1 or C/EBPα, documenting the role of LEF1 in the diminished ELA2 expression. We found that transduction of hematopoietic cells with LEF-1 cDNA resulted in the up-regulation of ELA2/NE synthesis, whereas inhibition of LEF-1 by shRNA led to a marked reduction in the levels of ELA2/NE. LEF-1 rescue of CD34+ cells isolated from 2 patients with CN resulted in granulocytic differentiation of the cells which was in line with increased levels of functionally active ELA2/NE.

Aberrant G-CSFR Signaling in Congenital Neutropenia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. SCI-45-SCI-45
Author(s):  
Julia Skokowa
Keyword(s):  
Transcription Factors ◽  
Myeloid Cells ◽  
Healthy Individuals ◽  
Granulocytic Differentiation ◽  
Congenital Neutropenia ◽  
Cyclic Neutropenia ◽  
Monosomy 7 ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Cd34 Cells

Abstract Clinical observations revealed that congenital neutropenia (CN) patients harboring either ELANE or HAX mutations have similar bone marrow morphology, responses to G-CSF therapy, requirements of G-CSF dosages, and the risk of developing leukemia. Therefore, we suggested a common pathomechanism of defective G-CSFR-triggered granulopoiesis downstream of both mutated genes in these patients. We identified severely diminished expression and functions of the transcription factors lymphoid enhancer binding factor-1 (LEF-1) and C/EBPa in myeloid cells of CN patients, in comparison to healthy individuals and patients with cyclic neutropenia (CyN). LEF-1 expression was abrogated in patients harboring either ELANE or HAX1 mutations, which suggested LEF-1 as a possible common candidate factor for defective G-CSFR signaling. We further identified a mechanism of the diminished LEF-1 expression downstream of HAX1 or ELANE mutations. HAX1 is HCLS1-Associated protein X1. HCLS1 is Hematopoietic Cell-Specific Lyn Substrate 1. We found that HCLS1 protein is expressed at high levels in human myeloid cells and is phosphorylated upon stimulation with G-CSF. HCLS1 interacted with LEF-1 protein, inducing nuclear translocation of LEF-1, LEF-1 autoregulation, C/EBPa activation, and granulocytic differentiation. In CN patients with HAX1 mutations, we found profound defects in the G-CSF-triggered phosphorylation of HCLS1, subsequently leading to abrogated nuclear transport and autoregulation of LEF-1. In CN patients with ELANE mutations we detected severely reduced levels of the natural inhibitor of neutrophil elastase (NE), and secretory leukocyte protease inhibitor (SLPI). We demonstrated the important role of SLPI in myeloid differentiation by activation of Erk1/2 phosphorylation and subsequent phErk1/2-triggered tyrosine phosphorylation and activation of the LEF-1 protein. Therefore, the direct link between ELANE mutations and diminished LEF-1 expression was established: in these patients LEF-1 protein expression is diminished due to the reduced levels of SLPI. We further evaluated how G-CSF treatment overcomes maturation arrest of granulopoiesis in CN patients despite the absence of LEF-1 and C/EBPa in myeloid cells. We identified nicotinamide phosphoribosyltransferase (NAMPT) as an essential enzyme mediating G-CSF-triggered granulopoiesis in healthy individuals and in CN patients. Treatment of healthy individuals with G-CSF resulted in upregulation of NAMPT levels in myeloid cells and in plasma. NAMPT and NAD+ amounts were even more dramatically elevated by G-CSF treatment of CN individuals. The molecular events triggered by NAMPT included elevation of NAD+, NAD+-dependent activation of protein deacetylase sirtuin-1 (SIRT1), binding of SIRT1 to the myeloid specific transcription factors C/EBPα and C/EBPβ, and activation of these transcription factors. In CN patients, C/EBPα expression is severely diminished; therefore “steady-state” granulopoiesis could not be activated. G-CSF treatment induces expression of C/EBPβ in these patients via NAMPT and SIRT1 and operated via the “emergency” pathway. We also investigated the patterns of acquisition of leukemia-associated-mutations in 31 CN patients developing leukemia using next-generation DNA deep sequencing. Intriguingly, 20 of the 31 patients (64.5%) demonstrated mutations within RUNX1. The majority of patients with RUNX1 mutations (85%) had acquired CSF3R mutations. Other leukemia-associated mutations in the patients with RUNX1 mutations were found infrequently. Cytogenetics of the leukemic cells revealed that 10 patients with RUNX1 mutations developed monosomy 7, and six patients had trisomy 21. Single cell analysis in two patients revealed that RUNX1 and CSF3R mutations were segregated in the same malignant clone. Functional studies demonstrated proliferative advantage of CD34+ cells transduced with mutated RUNX1 and CSF3R. By analysis of the leukemogenic role of the defective G-CSFR signaling in CN patients we identified a significant and sustained elevation in the levels of phospho-STAT5 in hematopoietic CD34+ cells of CN patients which were even higher in CN/ acute myeloid leukemia patients. The other possible reason for the leukemogenic transformation could be elevated NAMPT/SIRT-triggered deacetylation of tumor supressor protein p53, proto-oncogene FOXO3a and Akt proteins. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Heterozygous Mutations of Clpb As a Newly Identified and Frequent Cause of Severe Congenital Neutropenia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 433-433
Author(s):  
Julia T Warren ◽  
Peeradol Wattanasirakul ◽  
David H Spencer ◽  
Adam Locke ◽  
Vahagn Makaryan ◽  
...  
Keyword(s):  
Cord Blood ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Severe Congenital Neutropenia ◽  
Granulocytic Differentiation ◽  
Congenital Neutropenia ◽  
Original Cohort ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Chronic Neutropenia

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by severe chronic neutropenia from birth, premature death secondary to infectious complications, and transformation to myeloid malignancy. Although many cases of SCN are associated with mutations in ELANE, encoding the neutrophil elastase, roughly one-third of cases do not have an identifiable genetic cause. In collaboration with the Severe Chronic Neutropenia International Registry (SCNIR), we performed exome sequencing on 90 cases of congenital neutropenia. Heterozygous missense mutations of CLPB were identified in six patients with SCN. None of these patients had mutations in other genes known to cause SCN. A total of 5 different mutations were identified that clustered within the ATPase domain. Of note, all of these mutations were predicted to be functionally deleterious and had a frequency of <0.002% in the ExAC and gnomAD databases. We subsequently identified heterozygous CLPB mutations in an additional 3 cases of SCN that were not part of our original cohort. Prior studies showed that biallelic mutations of CLPB are associated with a syndrome defined by 3-methylglutaconic aciduria (3-MGA), cataracts, neurologic disease, and variable neutropenia. In our original cohort of 6 SCN patients with heterozygous CLPB mutations, 3-MGA was not present in the three cases where urine samples were available and none had reported cataracts. In total, 3 had neurologic abnormalities (2 seizures and 1 developmental delay). Of note, the CLPB mutations present in syndromic cases were distinct from those seen in our SCN cohort, and none of the mutations in our series are biallelic. CLPB encodes for caseinolytic peptidase B, a protein implicated in protein folding in bacteria and yeast but with an unknown role in human granulopoiesis. Based on these observations, we hypothesize that CLPB is required for normal basal granulopoiesis and that the heterozygous CLPB mutations identified in our study act in a dominant-negative fashion to disrupt granulopoiesis. To test this hypothesis, two complementary genetic approaches were employed. First, we used CRISPR-Cas9 gene editing to generate null mutations in CLPB in human cord blood-derived CD34+ hematopoietic stem/progenitor cells (HSPCs). Using this approach, we are able to achieve greater than 90% editing efficiency as assessed by next generation sequencing. The genetically modified HSPCs were cultured for 14 days under conditions that promote granulocytic differentiation. Modified HSPCs were also seeded into methylcellulose cultures to measure CFU-G. The percentage and absolute number of mature granulocytes, but not early granulocytic precursors (promyelocytes and myelocytes), were significantly reduced in cultures of gene-edited cord blood CD34+ cells. Moreover, the frequency of edited cells decreases over time in our culture system indicating that CLPB-knockout cells have a competitive disadvantage. A significant decrease in CFU-G also was observed. Second, we generated lentivirus expressing all 5 of the neutropenia-associated heterozygous CLPB mutations identified in our SCN cohort (N496K, E557K, R561G, R603H, and R620C). Expression of all of these mutants (except R603H) in cord blood-derived CD34+ cells was associated with a significant decrease in mature neutrophils and corresponding increase in early granulocytic precursors. These four CLPB mutants also resulted in a decrease in CFU-G. Collectively, these data strongly suggest that heterozygous mutations of CLPB are a new cause of congenital neutropenia. Indeed, in the North American population, CLPB mutations appear to be the second most common cause of congenital neutropenia, behind ELANE mutations. Studies are underway to examine the molecular mechanisms by which mutant CLPB disrupts granulopoiesis. Disclosures Dale: Coherus: Consultancy; Beheringer/Ingelheim: Consultancy; Athelas: Equity Ownership; Amgen: Consultancy, Research Funding; Sanofi Aventis: Consultancy, Honoraria; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Prolong: Consultancy; x4pharma: Consultancy, Honoraria, Research Funding.

LEF-1 Transcription Factor Regulates Proliferation and Differentiation of Myeloid Progenitors in Healthy Individuals and in Patients with Severe Congenital Neutropenia (CN).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 390-390 ◽  
Author(s):  
Julia Skokowa ◽  
Gunnar Cario ◽  
Vesna Bucan ◽  
Lan Dan ◽  
Zheng Wang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cyclin D1 ◽  
Target Genes ◽  
Healthy Controls ◽  
Severe Congenital Neutropenia ◽  
Healthy Individuals ◽  
Congenital Neutropenia ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Myeloid Progenitors

Abstract Wnt signaling pathway plays a key role in the signal transmission during cell growth, proliferation, differentiation and survival, especially in rapidly self-renewing tissues, such as bone marrow, skin, and intestinal mucosa. Regulation of target gene expression by Wnt signaling requires the presence of lymphoid enhancer factor 1 (LEF-1) and/or T-cell factors (TCFs) within the nucleus. Previously we reported 20 times lower or even absent expression of LEF-1 mRNA and protein in CD33+ myeloid progenitors from patients with severe congenital neutropenia (CN), as compared to healthy controls. Abolished LEF-1 expression in CD33+ progenitors from CN patients was accompanied by low levels of LEF-1 target genes, such as cyclin D1, c-myc, ELA2, and survivin. In the present study we aimed to characterize the role of LEF-1 transcription factor in myeloid cells from healthy individuals and from CN patients. Expression profile of LEF-1 mRNA during myeloid differentiation was measured by laser-assisted single-cell picking and real-time quantitative RT-PCR of different myeloid precursors (myeloblasts, promyelocytes, myelocytes/metamyelocytes, and mature granulocytes) from bone marrow smears of healthy controls and CN patients. LEF-1 mRNA was predominantly expressed in promyelocytes and myelocytes/metamyelocytes from healthy donors and was dramatically down-regulated in these cell types from CN patients. To investigate the functional consequence of LEF-1 downregulation, we specifically inhibitied LEF-1 expression in HL-60 promyelocytic cells and CD34+ cells from healthy individuals by lentiviral transduction of anti-LEF-1 shRNA. Interestingly, inhibition of LEF-1 resulted in down-regulation of LEF-1 target genes (cyclin D1, survivin, and c-myc), hematopoietic transcription factors C/EBPα and C/EBPε , and was accompanied by dramatically reduced proliferation and increased apoptosis in both HL-60 cells and CD34+ progenitors. Moreover, lentiviral transduction using vector containing LEF-1-GFP reporter into CD34+ cells from one CN patient led to normalization of expression of LEF-1 target genes (c-myc, survivin, cyclin D1), increased expression of C/EBPα , C/EBPε , and partial restoration of myelopoiesis. To investigate the cause of reduced LEF-1 expression in myeloid progenitors from CN patients, we sequenced the LEF-1 gene. We could not find any mutations in 15 CN patients studied. Therefore, regulatory mechanisms of LEF-1 expression, which are different from healthy controls, exist in CN. Interestingly, noggin, one of the activators of LEF-1 expression is significantly downregulated in CN patients. When exposed to noggin-containing medium, LEF-1 and LEF-1 target genes were induced in CD33+ cells from one CN patient, although the effect of noggin on myelopoiesis have to be analysed.Taken together, LEF-1 transcription factor regulate proliferation and differentiation of myeloid progenitors and is involved in the pathomechanism of CN.

scholarly journals LEF1 Enhances the Progression of Colonic Adenocarcinoma via Remodeling the Cell Motility Associated Structures

2021 ◽  
Vol 22 (19) ◽  
pp. 10870
Author(s):  
Li Xiao ◽  
Caixia Zhang ◽  
Xinyao Li ◽  
Chenshuang Jia ◽  
Lirong Chen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Molecular Mechanisms ◽  
Wnt Signaling Pathway ◽  
Colonic Adenocarcinoma ◽  
Mesenchymal Transition ◽  
Lamin B1 ◽  
Caco2 Cells ◽  
Binding Factor ◽  
Endothelial Mesenchymal Transition ◽  
Β Tubulin

Lymphoid enhancer-binding factor 1 (LEF1) is a key transcription factor mediating the Wnt signaling pathway. LEF1 is a regulator that is closely associated with tumor malignancy and is usually upregulated in cancers, including colonic adenocarcinoma. The underlying molecular mechanisms of LEF1 regulation for colonic adenocarcinoma progression remain unknown. To explore it, the LEF1 expression in caco2 cells was inhibited using an shRNA approach. The results showed that downregulation of LEF1 inhibited the malignancy and motility associated microstructures, such as polymerization of F-actin, β-tubulin, and Lamin B1 in caco2 cells. LEF1 inhibition suppressed the expression of epithelial/endothelial-mesenchymal transition (EMT) relevant genes. Overall, the current results demonstrated that LEF1 plays a pivotal role in maintaining the malignancy of colonic adenocarcinoma by remodeling motility correlated microstructures and suppressing the expression of EMT-relevant genes. Our study provided evidence of the roles LEF1 played in colonic adenocarcinoma progression, and suggest LEF1 as a potential target for colonic adenocarcinoma therapy.

scholarly journals Regulatory Role of microRNAs Targeting the Transcription Co-Factor ZNF521 in Normal Tissues and Cancers

2021 ◽  
Vol 22 (16) ◽  
pp. 8461
Author(s):  
Emanuela Chiarella ◽  
Annamaria Aloisio ◽  
Stefania Scicchitano ◽  
Heather Mandy Bond ◽  
Maria Mesuraca
Keyword(s):  
Transcription Factor ◽  
Molecular Mechanisms ◽  
Transitional Cell ◽  
Biological Functions ◽  
Aberrant Expression ◽  
Normal Tissues ◽  
Novel Mirna ◽  
Mirna Expression Profiling ◽  
Mirna Deregulation

Powerful bioinformatics tools have provided a wealth of novel miRNA–transcription factor networks crucial in controlling gene regulation. In this review, we focus on the biological functions of miRNAs targeting ZNF521, explaining the molecular mechanisms by which the dysregulation of this axis contributes to malignancy. ZNF521 is a stem cell-associated co-transcription factor implicated in the regulation of hematopoietic, neural, and mesenchymal stem cells. The aberrant expression of ZNF521 transcripts, frequently associated with miRNA deregulation, has been detected in several tumors including pancreatic, hepatocellular, gastric, bladder transitional cell carcinomas as well as in breast and ovarian cancers. miRNA expression profiling tools are currently identifying a multitude of miRNAs, involved together with oncogenes and TFs in the regulation of oncogenesis, including ZNF521, which may be candidates for diagnostic and prognostic biomarkers of cancer.

scholarly journals Endothelin 1-induced retinal ganglion cell death is largely mediated by JUN activation

2020 ◽  
Vol 11 (9) ◽  
Author(s):  
Olivia J. Marola ◽  
Stephanie B. Syc-Mazurek ◽  
Gareth R. Howell ◽  
Richard T. Libby
Keyword(s):  
Transcription Factor ◽  
Molecular Mechanisms ◽  
Ganglion Cells ◽  
Retinal Vessel ◽  
Vessel Diameter ◽  
Retinal Ganglion ◽  
Retinal Ganglion Cell Death ◽  
Ganglion Cell Death

Abstract Glaucoma is a neurodegenerative disease characterized by loss of retinal ganglion cells (RGCs), the output neurons of the retina. Multiple lines of evidence show the endothelin (EDN, also known as ET) system is important in glaucomatous neurodegeneration. To date, the molecular mechanisms within RGCs driving EDN-induced RGC death have not been clarified. The pro-apoptotic transcription factor JUN (the canonical target of JNK signaling) and the endoplasmic reticulum stress effector and transcription factor DNA damage inducible transcript 3 (DDIT3, also known as CHOP) have been shown to act downstream of EDN receptors. Previous studies demonstrated that JUN and DDIT3 were important regulators of RGC death after glaucoma-relevant injures. Here, we characterized EDN insult in vivo and investigated the role of JUN and DDIT3 in EDN-induced RGC death. To accomplish this, EDN1 ligand was intravitreally injected into the eyes of wildtype, Six3-cre+Junfl/fl (Jun−/−), Ddit3 null (Ddit3−/−), and Ddit3−/−Jun−/− mice. Intravitreal EDN1 was sufficient to drive RGC death in vivo. EDN1 insult caused JUN activation in RGCs, and deletion of Jun from the neural retina attenuated RGC death after EDN insult. However, deletion of Ddit3 did not confer significant protection to RGCs after EDN1 insult. These results indicate that EDN caused RGC death via a JUN-dependent mechanism. In addition, EDN signaling is known to elicit potent vasoconstriction. JUN signaling was shown to drive neuronal death after ischemic insult. Therefore, the effects of intravitreal EDN1 on retinal vessel diameter and hypoxia were explored. Intravitreal EDN1 caused transient retinal vasoconstriction and regions of RGC and Müller glia hypoxia. Thus, it remains a possibility that EDN elicits a hypoxic insult to RGCs, causing apoptosis via JNK-JUN signaling. The importance of EDN-induced vasoconstriction and hypoxia in causing RGC death after EDN insult and in models of glaucoma requires further investigation.

scholarly journals Bortezomib inhibits STAT5-dependent degradation of LEF-1, inducing granulocytic differentiation in congenital neutropenia CD34+ cells

Blood ◽  
2014 ◽  
Vol 123 (16) ◽  
pp. 2550-2561 ◽  
Author(s):  
Kshama Gupta ◽  
Inna Kuznetsova ◽  
Olga Klimenkova ◽  
Maksim Klimiankou ◽  
Johann Meyer ◽  
...  
Keyword(s):  
Granulocytic Differentiation ◽  
Congenital Neutropenia ◽  
Bortezomib Treatment ◽  
Cd34 Cells ◽  
Key Points

Key Points Hyperactivated STAT5a binds LEF-1 protein leading to NLK/NARF/ubiquitin-dependent degradation of LEF-1 followed by defective granulopoiesis. In CN patients, elevated levels of phospho-STAT5a resulted in diminished LEF-1 expression, which could be restored by bortezomib treatment.

Insights in the molecular mechanisms of an azole stress adapted laboratory-generated Aspergillus fumigatus strain

2021 ◽  
Author(s):  
Marion Aruanno ◽  
Samantha Gozel ◽  
Isabelle Mouyna ◽  
Josie E Parker ◽  
Daniel Bachmann ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Aspergillus Fumigatus ◽  
Molecular Mechanisms ◽  
Drug Transporters ◽  
Major Facilitator Superfamily ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Drug Transporter

Abstract Aspergillus fumigatus is the main cause of invasive aspergillosis, for which azole drugs are the first-line therapy. Emergence of pan-azole resistance among A. fumigatus is concerning and has been mainly attributed to mutations in the target gene (cyp51A). However, azole resistance may also result from other mutations (hmg1, hapE) or other adaptive mechanisms. We performed microevolution experiment exposing an A. fumigatus azole-susceptible strain (Ku80) to sub-minimal inhibitory concentration of voriconazole to analyze emergence of azole resistance. We obtained a strain with pan-azole resistance (Ku80R), which was partially reversible after drug relief, and without mutations in cyp51A, hmg1, and hapE. Transcriptomic analyses revealed overexpression of the transcription factor asg1, several ATP-binding cassette (ABC) and major facilitator superfamily transporters and genes of the ergosterol biosynthesis pathway in Ku80R. Sterol analysis showed a significant decrease of the ergosterol mass under voriconazole exposure in Ku80, but not in Ku80R. However, the proportion of the sterol compounds was similar between both strains. To further assess the role of transporters, we used the ABC transporter inhibitor milbemycine oxime (MLB). MLB inhibited transporter activity in both Ku80 and Ku80R and demonstrated some potentiating effect on azole activity. Criteria for synergism were reached for MLB and posaconazole against Ku80. Finally, deletion of asg1 revealed some role of this transcription factor in controlling drug transporter expression, but had no impact on azole susceptibility. This work provides further insight in mechanisms of azole stress adaptation and suggests that drug transporters inhibition may represent a novel therapeutic target. Lay Summary A pan-azole-resistant strain was generated in vitro, in which drug transporter overexpression was a major trait. Analyses suggested a role of the transporter inhibitor milbemycin oxime in inhibiting drug transporters and potentiating azole activity.

scholarly journals Progranulin modulates cartilage-specific gene expression via sirtuin 1–mediated deacetylation of the transcription factors SOX9 and P65

2020 ◽  
Vol 295 (39) ◽  
pp. 13640-13650 ◽  
Author(s):  
Dongxu Feng ◽  
Xiaomin Kang ◽  
Ruiqi Wang ◽  
He Chen ◽  
Kun Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Molecular Mechanisms ◽  
Sirtuin 1 ◽  
Cartilage Tissue ◽  
Nuclear Accumulation ◽  
Specific Gene ◽  
Causative Role ◽  
Human Cartilage ◽  
Cartilage Homeostasis

Progranulin (PGRN) is an autocrine growth factor that exerts crucial roles within cartilage tissue; however, the molecular mechanisms underlying PGRN-mediated cartilage homeostasis remain elusive. In the present study, we investigated the role of PGRN in regulating chondrocyte homeostasis and its therapeutic potential for managing osteoarthritis (OA). We found that PGRN levels are significantly increased in human cartilage in mild OA and that its expression is decreased in the cartilage in severe OA. In vitro, treatment of primary rat chondrocytes with recombinant PGRN significantly enhanced the levels of collagen type II α 1 chain (COL2A1) and aggrecan, and attenuated TNFα-induced up-regulation of matrix metallopeptidase 13 (MMP13) and ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5) in chondrocytes. These effects were abrogated in SIRT1−/− cells, indicating a causative role of SIRT1 in the effects of PGRN on protein expression in chondrocytes. Mechanistically, PGRN increased SIRT1 expression and activity, which reduced the acetylation levels of SRY-box transcription factor (SOX9) and transcription factor P65 (P65) and thereby promoted nuclear translocation of SOX9 and inhibited TNFα-induced P65 nuclear accumulation to maintain chondrocyte homeostasis. In conclusion, our findings reveal a mechanism of action for PGRN that maintains cartilage homeostasis and supports the notion that PGRN up-regulation may be a promising strategy for managing OA.

Sign in / Sign up

Export Citation Format

Share Document