Expression of Trk receptors in the developing mouse trigeminal ganglion: in vivo evidence for NT-3 activation of TrkA and TrkB in addition to TrkC

Development ◽  
1999 ◽  
Vol 126 (10) ◽  
pp. 2191-2203 ◽  
Author(s):  
E.J. Huang ◽  
G.A. Wilkinson ◽  
I. Farinas ◽  
C. Backus ◽  
K. Zang ◽  
...  
Keyword(s):  
Trigeminal Ganglion ◽  
Expression Patterns ◽  
Wild Type ◽  
Normal Numbers ◽  
Trk Receptors ◽  
Normal Number ◽  
Cell Counts ◽  
Trk Receptor ◽  
Almost All

Animals lacking neurotrophin-3 (NT-3) are born with deficits in almost all sensory ganglia. Among these, the trigeminal ganglion is missing 70% of the normal number of neurons, a deficit which develops during the major period of neurogenesis between embryonic stages (E) 10.5 and E13.5. In order to identify the mechanisms for this deficit, we used antisera specific for TrkA, TrkB, and TrkC to characterize and compare the expression patterns of each Trk receptor in trigeminal ganglia of wild type and NT-3 mutants between E10.5 and E15.5. Strikingly, TrkA, TrkB, and TrkC proteins appear to be exclusively associated with neurons, not precursors. While some neurons show limited co-expression of Trk receptors at E11.5, by E13. 5 each neuron expresses only one Trk receptor. Neuronal birth dating and cell counts show that in wild-type animals all TrkB- and TrkC-expressing neurons are generated before E11.5, while the majority of TrkA-expressing neurons are generated between E11.5 and E13.5. In mice lacking NT-3, the initial formation of the ganglion, as assessed at E10.5, is similar to that in wild-type animals. At E11.5, however, the number of TrkC-expressing neurons is dramatically reduced and the number of TrkC-immunopositive apoptotic profiles is markedly elevated. By E13.5, TrkC-expressing neurons are virtually eliminated. At E11.5, compared to wild type, the number of TrkB-expressing neurons is also reduced and the number of TrkB immunoreactive apoptotic profiles is increased. TrkA neurons are also reduced in the NT-3 mutants, but the major deficit develops between E12.5 and E13.5 when elevated numbers of TrkA-immunoreactive apoptotic profiles are detected. Normal numbers of TrkA- and TrkB-expressing neurons are seen in a TrkC-deficient mutant. Therefore, our data provide evidence that NT-3 supports the survival of TrkA-, TrkB- and TrkC-expressing neurons in the trigeminal ganglion by activating directly each of these receptors in vivo.

Transcriptional activation of the estrogen receptor

1993 ◽  
Vol 39 (2) ◽  
pp. 341-345 ◽  
Author(s):  
L L Wei
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Estrogen Receptors ◽  
Transcriptional Activation ◽  
Breast Cancer Cells ◽  
Wild Type ◽  
Resistant Phenotype ◽  
Estrogen Receptor Positive ◽  
Almost All

Abstract Almost all breast cancer tumors progress to a hormone-resistant state. Evidence is presented that the existence of mutant estrogen receptors may explain some hormone-resistant phenotypes. Breast tumor cells bearing a mutant receptor that is constitutively active and does not bind hormone would have unregulated cell growth and thus appear to be hormone-independent. Alternatively, breast cancer cells may contain estrogen receptors that are transcriptionally inactive but when co-expressed with wild-type receptors render normal estrogen receptors inactive. These cells would be considered estrogen receptor-positive but would be hormone-resistant. The hormone-resistant phenotype could be further complicated by the finding that other nonreceptor proteins may also modulate the transcriptional activity of estrogen receptors. These findings, if substantiated in vivo, could add to the complexity of the hormone-resistant phenotype. Different strategies of treatment will need to be developed to effectively treat the various subtypes of hormone-resistant breast tumors.

scholarly journals Myostatin Attenuation In Vivo Reduces Adiposity, but Activates Adipogenesis

Endocrinology ◽  
2016 ◽  
Vol 157 (1) ◽  
pp. 282-291 ◽  
Author(s):  
Naisi Li ◽  
Qiyuan Yang ◽  
Ryan G. Walker ◽  
Thomas B. Thompson ◽  
Min Du ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Muscle Mass ◽  
Wild Type ◽  
Cell Counts ◽  
Novel Approach ◽  
Brown Adipose ◽  
Nutrient Partitioning ◽  
Differentiation Marker

Abstract A potentially novel approach for treating obesity includes attenuating myostatin as this increases muscle mass and decreases fat mass. Notwithstanding, conflicting studies report that myostatin stimulates or inhibits adipogenesis and it is unknown whether reduced adiposity with myostatin attenuation results from changes in fat deposition or adipogenesis. We therefore quantified changes in the stem, transit amplifying and progenitor cell pool in white adipose tissue (WAT) and brown adipose tissue (BAT) using label-retaining wild-type and mstn−/− (Jekyll) mice. Muscle mass was larger in Jekyll mice, WAT and BAT mass was smaller and label induction was equal in all tissues from both wild-type and Jekyll mice. The number of label-retaining cells, however, dissipated quicker in WAT and BAT of Jekyll mice and was only 25% and 17%, respectively, of wild-type cell counts 1 month after induction. Adipose cell density was significantly higher in Jekyll mice and increased over time concomitant with label-retaining cell disappearance, which is consistent with enhanced expansion and differentiation of the stem, transit amplifying and progenitor pool. Stromal vascular cells from Jekyll WAT and BAT differentiated into mature adipocytes at a faster rate than wild-type cells and although Jekyll WAT cells also proliferated quicker in vitro, those from BAT did not. Differentiation marker expression in vitro, however, suggests that mstn−/− BAT preadipocytes are far more sensitive to the suppressive effects of myostatin. These results suggest that myostatin attenuation stimulates adipogenesis in vivo and that the reduced adiposity in mstn−/− animals results from nutrient partitioning away from fat and in support of muscle.

PI3K/Akt/FOXO3a Pathway Contributes to Thrombopoietin-Induced Proliferation of Primary Megakaryocytes In Vitro and In Vivo Via Modulation of p27Kip1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 202-202
Author(s):  
Takafumi Nakao ◽  
Amy E Geddis ◽  
Norma E. Fox ◽  
Kenneth Kaushansky
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Wild Type ◽  
Cycle Progression ◽  
P27kip1 Expression ◽  
Cell Counts ◽  
Deficient Mice

Abstract Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. In the previous study, we reported that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. The absence of PI3K activity results in a block of transition from G1 to S phase in these cells (Geddis AE et al. JBC2001276:34473–34479). However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. TPO induces phosphorylation of Akt and FOXO3a in both UT-7/TPO, a megakaryocytic cell line, and primary murine MKs in a PI3K dependent fashion. Cell cycle progression of UT-7/TPO cells is blocked in G1 phase by inhibition of PI3K. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in UT-7/TPO cells and primary MKs in a PI3K dependent fashion. UT-7/TPO stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to induce p27Kip1 expression after TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In an attempt to assess whether FOXO3a has an effect of MK proliferation in vivo, we compared the number of MKs in Foxo3a-deficient mice and in wild type controls. Although peripheral blood cell counts of erythrocytes, neutrophils, monocytes and platelets were normal in the Foxo3a-deficient mice, total nucleated marrow cell count of Foxo3a-deficient mice were 60% increased compared with wild type controls. In addition, the increase of MKs was more profound than that of total nucleated marrow cells; CD41+ MKs from Foxo3a-deficient mice increased 2.1-fold, and mature MKs with 8N and greater ploidy increased 2.5-fold, compared with wild type controls. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings strongly suggest that the effect of TPO on MK proliferation is mediated by PI3K/Akt-induced FOXO3a inactivation and subsequent p27Kip1 down-regulation in vitro and in vivo.

scholarly journals Blocking TLR2 in vivo Protects against Accumulation of Inflammatory Cells and Neuronal Injury in Experimental Stroke

2010 ◽  
Vol 31 (2) ◽  
pp. 757-766 ◽  
Author(s):  
Gina Ziegler ◽  
Dorette Freyer ◽  
Denise Harhausen ◽  
Uldus Khojasteh ◽  
Wilfried Nietfeld ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Neuronal Death ◽  
Focal Cerebral Ischemia ◽  
Neuronal Survival ◽  
Inflammatory Cells ◽  
Experimental Stroke ◽  
Wild Type ◽  
Antibody Treatment ◽  
Cell Counts

Reduced infarct volume in TLR2-knockout mice compared with C57Bl/6 wild-type mice has recently been shown in experimental stroke and confirmed in this study. We now also show a significant decrease of CD11b-positive cell counts and decreased neuronal death in the ischemic hemispheres of TLR2-deficient mice compared with C57Bl/6wt mice 2 days after transient focal cerebral ischemia. To examine the potential benefit of intravascular TLR2 inhibition, C57Bl/6wt mice were treated intraarterially with TLR2-blocking anti-TLR2 antibody (clone T2.5) after 45 minutes of cerebral ischemia and compared with control antibody (isotype) treated wild-type mice. Whereas T2.5-treated mice had no reduction in infarct volumes at 48 hours after reperfusion, they did have decreased numbers of CD11b-positive inflammatory cells and decreased neuronal death compared with isotype-treated control mice. Comparison of the isotype antibody treatment to control (saline) treatment showed no effects on infarct volumes or neuronal survival. However, mice treated with the control isotype antibody had increased numbers of CD11b-positive inflammatory cells compared with saline-treated animals. Thus, antibody treatment itself (i.e., control isotype antibody, but potentially of any antibody) may have adverse effects and limit therapeutic benefit of anti-TLR2-antibody therapy. We conclude that TLR2 mediates leukocyte and microglial infiltration and neuronal death, which can be attenuated by TLR2 inhibition. The TLR2 inhibition in vivo improves neuronal survival and may represent a future stroke therapy.

scholarly journals Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
Wenchao Feng ◽  
Dylan Minor ◽  
Mengyao Liu ◽  
Jinquan Li ◽  
Suzanne L. Ishaq ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Phase ◽  
Virulence Genes ◽  
Expression Patterns ◽  
Mouse Skin ◽  
Exponential Growth Phase ◽  
Wild Type ◽  
Subcutaneous Infection ◽  
Group A

ABSTRACT Group A Streptococcus (GAS) acquires mutations of the virulence regulator CovRS in human and mouse infections, and these mutations result in the upregulation of virulence genes and the downregulation of the protease SpeB. To identify in vivo mutants with novel phenotypes, GAS isolates from infected mice were screened by enzymatic assays for SpeB and the platelet-activating factor acetylhydrolase Sse, and a new type of variant that had enhanced Sse expression and normal levels of SpeB production was identified (the variants had a phenotype referred to as enhanced Sse activity [SseA+] and normal SpeB activity [SpeBA+]). SseA+ SpeBA+ variants had transcript levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an SseA+ SpeBA+ isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other SseA+ SpeBA+ isolates also had nonsense mutations or small indels in rocA. RocA and CovS mutants had similar levels of enhancement of the expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA but not mutations of CovS did not result in the downregulation of speB transcription at stationary growth phase or in subcutaneous infection of mice. GAS with RocA and CovS mutations caused greater enhancement of the expression of hasA than spyCEP in mouse skin infection than wild-type GAS did. RocA mutants ranked between wild-type GAS and CovS mutants in skin invasion, inhibition of neutrophil recruitment, and virulence in subcutaneous infection of mice. Thus, GAS RocA mutants can be selected in subcutaneous infections in mice and exhibit gene expression patterns and virulences distinct from those of CovS mutants. The findings provide novel information for understanding GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence.

Regulation of scute function by extramacrochaete in vitro and in vivo

Development ◽  
1994 ◽  
Vol 120 (12) ◽  
pp. 3595-3603 ◽  
Author(s):  
C.V. Cabrera ◽  
M.C. Alonso ◽  
H. Huikeshoven
Keyword(s):  
Expression Patterns ◽  
Transcription Activation ◽  
Loss Of Function ◽  
Wild Type ◽  
Helix Loop Helix ◽  
In The Wild ◽  
Yeast Assay ◽  
Number Of Cells

The pattern of adult sensilla in Drosophila is established by the dosage-sensitive interaction of two antagonistic groups of genes. Sensilla development is promoted by members of the achaete-scute complex and the daughterless gene whereas it is suppressed by whereas extramacrochaete (emc) and hairy. All these genes encode helix-loop-helix proteins. The products of the achaete-scute complex and daughterless interact to form heterodimers able to activate transcription. In this report, we show that (1) extra-macrochaete forms heterodimers with the achaete, scute, lethal of scute and daughterless products; (2) extramacrochaete inhibits DNA-binding of Achaete, Scute and Lethal of Scute/Daughterless heterodimers and Daughterless homodimers and (3) extramacrochaete inhibits transcription activation by heterodimers in a yeast assay system. In addition, we have studied the expression patterns of scute in wild-type and extramacrochaete mutant imaginal discs. Expression of scute RNA during imaginal development occurs in groups of cells, but high levels of protein accumulate in the nuclei of only a subset of the RNA-expressing cells. The pattern is dynamic and results in a small number of protein-containing cells that correspond to sensillum precursors. extramacrochaete loss-of-function alleles develop extra sensilla and correspondingly display a larger number of cells with scute protein. These cells appear to arise from those that in the wild type already express scute RNA; hence, extramacrochaete is a repressor of scute function whose action may take place post-transcriptionally.

JAK2 V617F Mutation Induces a Myeloproliferative Disorder in Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 376-376
Author(s):  
Thomas G.P. Bumm ◽  
Collin Elsea ◽  
Lisa G. Wood ◽  
Daniel W. Sherbenou ◽  
Ian J. Griswold ◽  
...  
Keyword(s):  
Pearson Correlation ◽  
Jak2 V617f ◽  
Peripheral Blood Smear ◽  
P Value ◽  
Wild Type ◽  
Empty Vector ◽  
Cell Counts ◽  
Prolonged Culture

Abstract Background: An activating mutation (V617F) in the JH2 domain of the Jak2 kinase is found in 74%–97% of patients with polycythemia vera (PV), 23%–57% of patients with essential thrombocythemia and 35%–57% of patients with idiopathic myelofibrosis. We studied the effects of this mutant in murine hematopoietic cells in vitro and in vivo. Materials and Methods: Murine wild type (Jak2-WT) and V617F mutant JAK2 (Jak2-V617F) were cloned into the MIGR1-IRES-GFP retroviral vector. BaF3 cells stably expressing mutant and wild type Jak2 were generated by electroporation followed by prolonged culture in IL-3 containing media and FACS selection for GFP-positive cells. Proliferation and viability assays were performed in graded concentrations of IL-3. Bone marrow from 5-FU treated Balb/c mice were infected with JAK2-V617F, JAK2-WT and empty vector retrovirus and injected into lethally irradiated recipients. The mice were monitored by observation and blood counts. Plasma levels of erythropoietin (EPO) were determined by ELISA on day 44 after transplantation. 1-way analysis of variance (ANOVA) was used to compare differences between cell line and hematologic parameters. The relationship between EPO levels and Hct/Hgb was analysed by Pearson correlation. Results: BaF3 cells expressing JAK2-V617F showed enhanced proliferation in response to murine IL-3 compared to cells expressing Jak2-WT (p<0.001) and parental cells (p= 0.009). Similarly, there was a trend for increased viability (p=0.071 vs. Jak-WT and p=0.059 vs. parental cells). Sudden complete IL-3 withdrawal induced >95% cell death, followed by outgrowth of IL-3-independent lines after 20 days. Mice transplanted with Jak2-V617F showed increased median hematocrit (Hct), hemoglobin (Hgb) and median corpuscular volume (MCV) compared to Jak2-WT and empty vector mice (table). There was a trend for increased white cell counts (WBC). Median EPO levels were 65 pg/ml in the Jak2-V617F mice compared to 88 pg/ml in the Jak2-WT and 131 pg/ml in the empty vector controls (p= 0.032). There was a significant correlation between EPO and Hct (p = 0.011, r = −0.677) and Hgb (p= 0.002, r= −0.770) across the three groups of mice. On day 44 post transplant one Jak2-V617F mouse developed trilineage MPD with a Hct of 56.8%, leukocytosis (22.6 x 103/ul) and thrombocytosis (1004 x 103/ul). The peripheral blood smear showed neutrophilia, elevated platelets and multiple nucleated red cells. Necropsy revealed hepatosplenomegaly. Conclusions: (i) JAK2-V617F induces hypersensitivity to IL-3 in BaF3 cells. Complete IL-3 independence was seen only after “crisis” and prolonged culture, suggesting additional mutations may be required. (ii) In a murine transduction-transplantation model JAK2-V617F induces MPD closely mimicking PV. Studies are in progress to determine whether additional phenotypes may be observed in a larger cohort of animals. JAK2-V617F (n= 4) JAK2- WT (n= 4) MIGR-1 vector-control (n= 5) P value (V617F vs. WT) P value (V617F vs. empty vector) Median blood parameters (range) in groups of mice 44 days after transplant Hct (%) 51.1 (44.6–56.8) 43.7 (42.8–45) 43.6 (40.4–45.2) P = 0.015 P= 0.011 Hgb (g/dl) 16.2 (14.6–17.6) 14.6 (14.2–15.4) 14.2 (13–15) P= 0.033 P= 0.010 MCV (fl) 49.5 (47–54) 46.2 (46–47) 46.2 (45–48) P= 0.041 P= 0.031 WBC (103/ul) 9.6 (3.8–22.6) 5.5 (3.6–9.4) 3.4 (2.2–4.6) ns P= 0.096 EPO (pg/ml) 65 (44–98) 88 (80–112) 131 (95–231) ns P= 0.032

scholarly journals AprlMutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

2014 ◽  
Vol 197 (5) ◽  
pp. 932-942 ◽  
Author(s):  
Juliana Durack ◽  
Thomas P. Burke ◽  
Daniel A. Portnoy
Keyword(s):  
Listeria Monocytogenes ◽  
Atp Hydrolysis ◽  
Colony Morphology ◽  
Wild Type ◽  
Swarming Motility ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Culture Cells ◽  
Almost All

The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogenListeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence inL. monocytogenes; however, the mechanism of export via this pathway is poorly understood.L. monocytogenessecA2mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility toL. monocytogenessecA2mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by thelmo2769operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation insecYwas identified that conferred smooth colony morphology tosecA2mutants, restored wild-type plaque formation, and increased virulence in mice. ThissecYmutation resembled aprlsuppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although theprlmutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1mutant was still less virulentin vivothan the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.

scholarly journals MiR858b Inhibits Proanthocyanidin Accumulation by the Repression of DkMYB19 and DkMYB20 in Persimmon

2020 ◽  
Vol 11 ◽  
Author(s):  
Sichao Yang ◽  
Meng Zhang ◽  
Liqing Xu ◽  
Zhengrong Luo ◽  
Qinglin Zhang
Keyword(s):  
Expression Patterns ◽  
Transient Transformation ◽  
Structural Genes ◽  
Wild Type ◽  
Rna Ligase ◽  
Key Factor ◽  
R2r3 Myb ◽  
Posttranscriptional Level

Persimmon proanthocyanidin (PA) biosynthesis is controlled by structural genes and regulated by transcription factors (TFs). MicroRNAs are a key factor involved in regulating gene expression at the posttranscriptional level whose functions in persimmon PA biosynthesis are poorly understood. Here, we identified a microRNA, miR858b, that putatively targets two R2R3-MYB TFs, DkMYB19 and DkMYB20. DkMYB19, DkMYB20, and miR858b showed divergent expression patterns during fruit development, and the interaction between miR858b and DkMYB19 or DkMYB20 was experimentally validated by 5′ RNA ligase-mediated RACE, LUC enzyme activity analysis, and GFP signal detection. The DkMYB19 localized to the nucleus as well as the cytoplasm and DkMYB20 localized to the nucleus. The overexpression of miR858b led to the downregulation of DkMYB19 and DkMYB20, which reduced the content of PA, whereas a reduction in miR858b activity upregulated DkMYB19 and DkMYB20, resulting in a high content of PA in leaves transiently expressing a small tandem target mimic construct for blocking miR858 (STTM858b) in vivo. The transient transformation of miR858b in fruit discs in vitro also reduced the content of PA, while the content of PA increased under the transient transformation of fruit discs with STTM858b, DkMYB19, or DkMYB20. A similar phenomenon was observed upon the overexpression of miR858b in wild-type (WT) Arabidopsis and DkMYB19 or DkMYB20 in persimmon leaf calli. These findings suggested that miR858b repressed the expression of DkMYB19 and DkMYB20, which contributed to the PA accumulation in persimmon.

scholarly journals KLF3 Represses the Inflammatory Response in Macrophages

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 36-36
Author(s):  
Jessica M Salmon ◽  
Casie Leigh Reed ◽  
Maddyson Bender ◽  
Helen Lorraine Mitchell ◽  
Vanessa Fox ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Time Course ◽  
Target Genes ◽  
Wild Type ◽  
Cell Counts ◽  
Inflammatory Macrophages

Krüppel-like factors (KLFs) are a family of transcription factors that play essential roles in the development and differentiation of the hematopoietic system. These transcription factors possess highly conserved C-terminal zinc-finger motifs, which enable their binding to GC-rich, or CACC-box, motifs in promoter and enhancer regions of target genes. The N-terminal domains of these proteins are more varied and mediate the recruitment of various co-factors, which can form a complex with either activator or repressor function. Acting primarily as a gene repressor through its recruitment of CtBPs and histone deacetylases (HDACs) [1], we have recently shown that KLF3 competes with KLF1 bound sites in the genome to repress gene expression during erythropoiesis [2]. However, the function of Klf3 in other lineages has been less well studied. This widely expressed transcription factor has reported roles in the differentiation of marginal zone B cells, eosinophil function and inflammation [3]. We utilised the Klf3-null mouse model [4] to more closely examine the role of Klf3 in innate inflammatory cells. These mice exhibit elevated white cell counts, including monocytes (Figure 1A), and inflammation of the skin. Conditional knockout of Klf4 in myeloid cells leads to a deficiency of inflammatory macrophages [5]. To test our hypothesis KLF3 normally represses inflammation, perhaps by antagonising the action of KLF4, bone-marrow derived macrophages (BMDM) were generated from wild-type or Klf3-null mice and stimulated with the bacterial toxin lipopolysaccharide (LPS). In wild type BMDM, LPS induces Klf3 gene expression and activation then delayed repression of target genes such as Lgals3 (galectin-3) over a 21 hour time course (Figure 1B). Quantitative real-time PCR and mRNA-seq of WT v Klf3-null macrophages identified ~100 differentially expressed genes involved in proliferation, macrophage activation and inflammation. We transduced the monocyte cell line, RAW264.7 (that expresses Klf4, Klf3 and Klf2), with a retroviral vector expressing a tamoxifen-inducible KLF3-ER fusion construct. KLF3 induced cell cycle arrest and macrophage differentiation. We will report on KLF3-induced gene expression changes (repression and activation), and ChIP-seq for KLF3, in RAW cells. The results shed light on the mechanism by which KLF3 normally represses monocyte/macrophage responses to infection. This study highlights the importance of key transcriptional regulators that tightly control gene expression during inflammation. Loss of Klf3 leads to alterations in this process, resulting in hyper-activation of inflammatory macrophages, increased white cell counts and inflammation of the skin. A greater knowledge of the inflammatory process and how it is regulated is important for our understanding of acute infection and inflammatory disease. Further studies are planned to investigate the role of the KLF3 transcription factor in response to inflammation in vivo. References: 1. Pearson, R., et al., Kruppel-like transcription factors: A functional family. Int J Biochem Cell Biol, 2007. W2. Ilsley, M.D., et al., Kruppel-like factors compete for promoters and enhancers to fine-tune transcription. Nucleic Acids Res, 2017. 45(11): p. 6572-6588. W3. Knights, A.J., et al., Kruppel-like factor 3 (KLF3) suppresses NF-kappaB-driven inflammation in mice. J Biol Chem, 2020. 295(18): p. 6080-6091. W4. Sue, N., et al., Targeted disruption of the basic Kruppel-like factor gene (Klf3) reveals a role in adipogenesis. Mol Cell Biol, 2008. 28(12): p. 3967-78. W5. Alder, J.K., et al., Kruppel-like factor 4 is essential for inflammatory monocyte differentiation in vivo. J Immunol, 2008. 180(8): p. 5645-52. Figure 1: Elevated WCC (A) and inflammatory markers (B) in BMDM after LPS stimulation. 1. Total WCC in adult mice (3-6 months old) of the indicated genotypes. There is a statistically significant increase in the WCC in Klf3-/- v wild type mice (P<0.001 by student's t test). B. Time course (hours) after LPS stimulation of confluent BMDM. Klf3 is induced 3-fold by LPS and KLF3-target genes such as Lgals3 are not fully repressed by 21 hours in knockout mice. Figure 1 Disclosures Perkins: Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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