Project description:Ets-2 is a ubiquitous transcription factor activated after phosphorylation at threonine-72. Previous studies highlighted the importance of phosphorylated ets-2 in lung inflammation and extracellular matrix remodeling, two pathways involved in pulmonary fibrosis. We hypothesized that phosphorylated ets-2 played an important role in pulmonary fibrosis, and we sought to determine the role of ets-2 in its pathogenesis. We challenged ets-2 (A72/A72) transgenic mice (harboring a mutated form of ets-2 at phosphorylation site threonine-72) and ets-2 (wild-type/wild-type [WT/WT]) control mice with sequential intraperitoneal injections of bleomycin, followed by quantitative measurements of lung fibrosis and inflammation and primary cell in vitro assays. Concentrations of phosphorylated ets-2 were detected via the single and dual immunohistochemical staining of murine lungs and lung sections from patients with idiopathic pulmonary fibrosis. Ets-2 (A72/A72) mice were protected from bleomycin-induced pulmonary fibrosis, compared with ets-2 (WT/WT) mice. This protection was characterized by decreased lung pathological abnormalities and the fibrotic gene expression of Type I collagen, Type III collagen, α-smooth muscle actin, and connective tissue growth factor. Immunohistochemical staining of lung sections from bleomycin-treated ets-2 (WT/WT) mice and from patients with idiopathic pulmonary fibrosis demonstrated increased staining of phosphorylated ets-2 that colocalized with Type I collagen expression and to fibroblastic foci. Lastly, primary lung fibroblasts from ets-2 (A72/A72) mice exhibited decreased expression of Type I collagen in response to stimulation with TGF-β, compared with fibroblasts from ets-2 (WT/WT) mice. These data indicate the importance of phosphorylated ets-2 in the pathogenesis of pulmonary fibrosis through the expression of Type I collagen and (myo)fibroblast activation.
Project description:KLF8 (Krüppel-like factor 8) is a zinc-finger transcription factor known to play an essential role in the regulation of the cell cycle, apoptosis, and differentiation. However, its physiological roles and functions in adipogenesis remain unclear. In the present study, we show that KLF8 acts as a key regulator controlling adipocyte differentiation. In 3T3-L1 preadipocytes, we found that KLF8 expression was induced during differentiation, which was followed by expression of peroxisome proliferator-activated receptor ? (PPAR?) and CCAAT/enhancer-binding protein ? (C/EBP?). Adipocyte differentiation was significantly attenuated by the addition of siRNA against KLF8, whereas overexpression of KLF8 resulted in enhanced differentiation. Furthermore, luciferase reporter assays demonstrated that overexpression of KLF8 induced PPAR?2 and C/EBP? promoter activity, suggesting that KLF8 is an upstream regulator of PPAR? and C/EBP?. The KLF8 binding sites were localized by site mutation analysis to -191 region in C/EBP? promoter and -303 region in PPAR? promoter, respectively. Taken together, these data reveal that KLF8 is a key component of the transcription factor network that controls terminal differentiation during adipogenesis.
Project description:Milk fat globule-EGF factor 8 (MFG-E8) is a secreted glycoprotein that regulates tissue homeostasis, possesses potent anti-inflammatory properties, and protects against tissue injury. The human pancreas expresses MFG-E8; however, the role of MFG-E8 in the pancreas remains unclear. We examined the expression of MFG-E8 in the pancreas at baseline and during cerulein-induced acute pancreatitis in mice and determined whether MFG-E8 attenuates the progression of pancreatitis, a serious inflammatory condition that can be life-threatening. We administered cerulein to wild-type (WT) and Mfge8 knockout (KO) mice to induce pancreatitis. Immunoblot analysis showed that MFG-E8 is constitutively expressed in the murine pancreas and is increased in mice with cerulein-induced acute pancreatitis. In situ hybridization revealed that ductal epithelial cells in the mouse pancreas express Mfge8 transcripts at baseline. During pancreatitis, Mfge8 transcripts were abundantly expressed in acinar cells and endothelial cells in addition to ductal epithelial cells. Knocking out Mfge8 in mice exacerbated the severity of cerulein-induced acute pancreatitis and delayed its resolution. In contrast, administration of recombinant MFG-E8 attenuated cerulein-induced acute pancreatitis and promoted repair of pancreatic injury in Mfge8 KO mice. Taken together, our study suggests that MFG-E8 protects the pancreas against inflammatory injury and promotes pancreatic tissue repair. MFG-E8 may represent a novel therapeutic target in acute pancreatitis.
Project description:AimsEndothelial colony forming cells (ECFCs) participate in post-natal vasculogenesis. We previously reported that vascular endothelial growth factor (VEGF) promotes human ECFC differentiation through AMP-activated protein kinase (AMPK) activation. However, the mechanisms underlying transcriptional regulation of ECFC differentiation still remain largely elusive. Here, we investigated the role of transcription factor Krüppel-like factor 2 (KLF2) in the regulation of ECFC function.Methods and resultsHuman ECFCs were isolated from cord blood and cultured. Treatment with VEGF significantly increased endothelial markers in ECFCs and their capacity for migration and tube formation. The mRNA and protein levels of KLF2 were also significantly up-regulated. This up-regulation was abrogated by AMPK inhibition or by knockdown of KLF2 with siRNA. Furthermore, adenovirus-mediated overexpression of KLF2 promoted ECFC differentiation by enhancing expression of endothelial cell markers, reducing expression of progenitor cell markers, and increasing the capacity for tube formation in vitro, indicating the important role of KLF2 in ECFC-mediated angiogenesis. Histone deacetylase 5 (HDAC5) was phosphorylated by AMPK activity induced by VEGF and the AMPK agonist AICAR (5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide). In vivo angiogenesis assay revealed that overexpression of KLF2 in bone-marrow-derived pro-angiogenic progenitor cells promoted vessel formation when the cells were implanted in C57BL/6 mice.ConclusionUp-regulation of KLF2 by AMPK activation constitutes a novel mechanism of ECFC differentiation, and may have therapeutic value in the treatment of ischaemic heart disease.
Project description:BackgroundCanine visceral hemangiosarcoma (HSA) is a highly aggressive cancer of endothelial origin that closely resembles visceral angiosarcoma in humans, both clinically and histopathologically. Currently there is an unmet need for new diagnostics and therapies for both forms of this disease. The goal of this study was to utilize Chromatin run-on sequencing (ChRO-seq) and immunohistochemistry (IHC) to identify gene and protein expression signatures that may be important drivers of HSA progression.ResultsChRO-seq was performed on tissue isolated from 17 HSA samples and 4 normal splenic samples. Computational analysis was then used to identify differentially expressed genes and these factors were subjected to gene ontology analysis. ChRO-seq analysis revealed over a thousand differentially expressed genes in HSA tissue compared with normal splenic tissue (FDR < 0.005). Interestingly, the majority of genes overexpressed in HSA tumor tissue were associated with extracellular matrix (ECM) remodeling. This observation correlated well with our histological analysis, which found that HSA tumors contain a rich and complex collagen network. Additionally, we characterized the protein expression patterns of two highly overexpressed molecules identified in ChRO-seq analysis, podoplanin (PDPN) and laminin alpha 4 (LAMA4). We found that the expression of these two ECM-associated factors appeared to be largely limited to transformed endothelial cells within the HSA lesions.ConclusionOutcomes from this study suggest that ECM remodeling plays an important role in HSA progression. Additionally, our study identified two potential novel biomarkers of HSA, PDPN and LAMA4. Interestingly, given that function-blocking anti-PDPN antibodies have shown anti-tumor effects in mouse models of canine melanoma, our studies raise the possibility that these types of therapeutic strategies could potentially be developed for treating canine HSA.
Project description:The binding of tumor necrosis factor-like cytokine 1A (TL1A) to death receptor 3 (DR3) plays an important role in the interaction between dendritic cells (DCs) and T cells and contributes to intestinal inflammation development. However, the mechanism by which DCs expressing TL1A mediate helper T (Th) cell differentiation in the intestinal lamina propria (LP) during the pathogenesis of inflammatory bowel disease remains unclear. In this study, we found that TL1A/DR3 promoted Th1 and Th17 cell differentiation in T-T and DC-T cell interaction-dependent manners. TL1A-deficient CD4+ T cells failed to polarize into Th1/Th17 cells and did not cause colonic inflammation in a T cell transfer colitis model. Notably, TL1A was located in the cytoplasm and nuclei of DCs, positively regulated the DC-specific ICAM-grabbing nonintegrin/RAF1/nuclear factor κB signaling pathway, enhanced the antigen uptake ability of DCs, and promoted TLR4-mediated DC activation, inducing naive CD4+ T cell differentiation into Th1 and Th17 cells. Our work reveals that TL1A plays a regulatory role in inflammatory bowel disease pathogenesis.
Project description:ASXL1 mutations are found in a spectrum of myeloid malignancies with poor prognosis. Recently, we reported that Asxl1(+/-) mice develop myelodysplastic syndrome (MDS) or MDS and myeloproliferative neoplasms (MPN) overlapping diseases (MDS/MPN). Although defective erythroid maturation and anemia are associated with the prognosis of patients with MDS or MDS/MPN, the role of ASXL1 in erythropoiesis remains unclear. Here, we showed that chronic myelomonocytic leukemia (CMML) patients with ASXL1 mutations exhibited more severe anemia with a significantly increased proportion of bone marrow (BM) early stage erythroblasts and reduced enucleated erythrocytes compared to CMML patients with WT ASXL1. Knockdown of ASXL1 in cord blood CD34(+) cells reduced erythropoiesis and impaired erythrocyte enucleation. Consistently, the BM and spleens of VavCre(+);Asxl1(f/f) (Asxl1(∆/∆)) mice had less numbers of erythroid progenitors than Asxl1(f/f) controls. Asxl1(∆/∆) mice also had an increased percentage of erythroblasts and a reduced erythrocyte enucleation in their BM compared to littermate controls. Furthermore, Asxl1(∆/∆) erythroblasts revealed altered expression of genes involved in erythroid development and homeostasis, which was associated with lower levels of H3K27me3 and H3K4me3. Our study unveils a key role for ASXL1 in erythropoiesis and indicates that ASXL1 loss hinders erythroid development/maturation, which could be of prognostic value for MDS/MPN patients.
Project description:Streptomyces griseus S4-7 was originally isolated from the strawberry rhizosphere as a microbial agent responsible for Fusarium wilt suppressive soils. S. griseus S4-7 shows specific and pronounced antifungal activity against Fusarium oxysporum f. sp. fragariae. In the Streptomyces genus, the whi transcription factors are regulators of sporulation, cell differentiation, septation, and secondary metabolites production. wblE2 function as a regulator has emerged as a new group in whi transcription factors. In this study, we reveal the involvement of the wblE2 transcription factor in the plant-protection by S. griseus S4-7. We generated ΔwblE, ΔwblE2, ΔwhiH, and ΔwhmD gene knock-out mutants, which showed less antifungal activity both in vitro and in planta. Among the mutants, wblE2 mutant failed to protect the strawberry against the Fusarium wilt pathogen. Transcriptome analyses revealed major differences in the regulation of phenylalanine metabolism, polyketide and siderophore biosynthesis between the S4-7 and the wblE2 mutant. The results contribute to our understanding of the role of streptomycetes wblE2 genes in a natural disease suppressing system.
Project description:Actin filaments are essential for plant adaptation to high temperatures. However, the molecular mechanisms of actin filaments in plant thermal adaptation remain unclear. Here, we found that the expression of Arabidopsis actin depolymerization factor 1 (AtADF1) was repressed by high temperatures. Compared with wild-type seedlings (WT), the mutation of AtADF1 and the overexpression of AtADF1 led to promoted and inhibited plant growth under high temperature conditions, respectively. Further, high temperatures induced the stability of actin filaments in plants. Compared with WT, Atadf1-1 mutant seedlings showed more stability of actin filaments under normal and high temperature conditions, while the AtADF1 overexpression seedlings showed the opposite results. Additionally, AtMYB30 directly bound to the promoter of AtADF1 at a known AtMYB30 binding site, AACAAAC, and promoted the transcription of AtADF1 under high temperature treatments. Genetic analysis further indicated that AtMYB30 regulated AtADF1 under high temperature treatments. Chinese cabbage ADF1 (BrADF1) was highly homologous with AtADF1. The expression of BrADF1 was inhibited by high temperatures. BrADF1 overexpression inhibited plant growth and reduced the percentage of actin cable and the average length of actin filaments in Arabidopsis, which were similar to those of AtADF1 overexpression seedlings. AtADF1 and BrADF1 also affected the expression of some key heat response genes. In conclusion, our results indicate that ADF1 plays an important role in plant thermal adaptation by blocking the high-temperature-induced stability of actin filaments and is directly regulated by MYB30.
Project description:Virus-induced gene silencing (VIGS) is a common reverse genetics strategy for characterizing the function of genes in plants. The detailed mechanism governing RNA silencing efficiency triggered by viruses is largely unclear. Here, we reveal that a petunia (Petunia hybrida) ocs element binding factor, PhOBF1, one of the basic leucine zipper (bZIP) transcription factors, was up-regulated by Tobacco rattle virus (TRV) infection. Simultaneous silencing of PhOBF1 and a reporter gene, phytoene desaturase (PDS) or chalcone synthase (CHS), by TRV-based VIGS led to a failure of the development of leaf photobleaching or the white-corollas phenotype. PhOBF1 silencing caused down-regulation of RNA silencing-related genes, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonautes (AGOs). After inoculation with the TRV-PhPDS, PhOBF1-RNAi lines exhibited a substantially impaired PDS silencing efficiency, whereas overexpression of PhOBF1 resulted in a recovery of the silencing phenotype (photobleaching) in systemic leaves. A compromised resistance to TRV and Tobacco mosaic virus was found in PhOBF1-RNAi lines, while PhOBF1-overexpressing lines displayed an enhanced resistance to their infections. Compared with wild-type plants, PhOBF1-silenced plants accumulated lower levels of free salicylic acid (SA), salicylic acid glucoside, and phenylalanine, contrarily to higher levels of those in plants overexpressing PhOBF1. Furthermore, transcripts of a number of genes associated with the shikimate and phenylpropanoid pathways were decreased or increased in PhOBF1-RNAi or PhOBF1-overexpressing lines, respectively. Taken together, the data suggest that PhOBF1 regulates TRV-induced RNA silencing efficiency through modulation of RDRs, DCLs, and AGOs mediated by the SA biosynthesis pathway.