scholarly journals Inhibition of miR-665 alleviates lipopolysaccharide-induced inflammation via up-regulation of SOCS7 in chondrogenic ATDC5 cells

2020 ◽  
Vol 19 (10) ◽  
pp. 2067-2072
Author(s):  
Qiaoyi Ning ◽  
Yiting He ◽  
Wukai Ma ◽  
Fang Tang ◽  
Ying Huang ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Luciferase Reporter ◽  
Cytokine Signaling ◽  
Necrosis Factor Alpha ◽  
Inflammatory Injury ◽  
Expression Levels ◽  
Over Expression ◽  
Qrt Pcr ◽  
Lps Treatment

Purpose: To examine the effect and mechanism of action of miR-665 in osteoarthritis.Methods: An in vitro inflammatory injury model of osteoarthritis was established using chondrogenic ATDC5 cells with lipopolysaccharide (LPS) treatment. The expression levels of inflammatory cytokines were determined by enzyme-linked immunosorbent assays (ELISAs) and by quantitative real-time polymerase chain reaction (qRT-PCR). A binding target for miR-665 was predicted using TargetScan and then evaluated using a dual-luciferase reporter assay.Results: Treatment with LPS significantly up-regulated the inflammatory cytokine expressions of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α), in ATDC5 cells (p < 0.01), and the expression of miRNA-665 was significantly increased in LPS-treated ATDC5 cells (p < 0.01).Knockdown of miR-665 down-regulated the expression levels of these inflammatory cytokines. Suppressor of cytokine signaling 7 (SOCS7) was identified as a target of miR-665. Data from qRT-PCR and western-blot analyses indicated that SOCS7 expression was promoted by miR-665  inhibition and inhibited by miR-665 over-expression. LPS treatment significantly decreased the expression of SOCS7 protein in ATDC5 cells (p < 0.01), and over-expression of SOCS7 attenuated the LPS-stimulated inflammatory injury. In addition, over-expression of miR-655 enhanced the inflammatory injury and reversed the protective effect of SOCS7 against LPS-stimulated inflammation.Conclusion: Inhibition of miR-665 alleviated LPS-stimulated inflammatory injury in ATDC5 cells via the up-regulation of SOCS7, suggesting a potential therapeutic target for osteoarthritis. Keywords: MiR-665, Lipopolysaccharide, Inflammation, SOCS7, Chondrogenic, ATDC5

scholarly journals Anti-inflammatory activity of diindolylmethane alleviates Riemerella anatipestifer infection in ducks

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242198
Author(s):  
Cherry P. Fernandez-Colorado ◽  
Paula Leona T. Cammayo ◽  
Rochelle A. Flores ◽  
Binh T. Nguyen ◽  
Woo H. Kim ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Inflammatory Diseases ◽  
Splenic Lymphocytes ◽  
Expression Levels ◽  
Riemerella Anatipestifer ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Anti Inflammatory

3,3’-Diindolylmethane (DIM) is found in cruciferous vegetables and is used to treat various inflammatory diseases because of its potential anti-inflammatory effects. To investigate effects of DIM in Riemerella anatipestifer-infected ducks which induce upregulation of inflammatory cytokines, ducks were treated orally with DIM at dose of 200 mg/kg/day and infected the following day with R. anatipestifer. Infected and DIM-treated ducks exhibited 14% increased survival rate and significantly decreased bacterial burden compared to infected untreated ducks. Next, the effect on the expression level of inflammatory cytokines (interleukin [IL]-17A, IL-17F, IL-6, IL-1β) of both in vitro and in vivo DIM-treated groups was monitored by quantitative reverse-transcription PCR (qRT-PCR). Generally, the expression levels of the cytokines were significantly reduced in DIM-treated splenic lymphocytes stimulated with killed R. anatipestifer compared to stimulated untreated splenic lymphocytes. Similarly, the expression levels of the cytokines were significantly reduced in the spleens and livers of DIM-treated R. anatipestifer–infected ducks compared to infected untreated ducks. This study demonstrated the ameliorative effects of DIM in ducks infected with R. anatipestifer. Thus, DIM can potentially be used to prevent and/or treat R. anatipestifer infection via inhibition of inflammatory cytokine expression.

scholarly journals LncRNA SNHG22 Sponges miR-128-3p to Promote Progression of Colorectal Cancer through Upregulating E2F3

2020 ◽  
Author(s):  
Yao Jianning ◽  
Wang Chunfeng ◽  
Dong Xuyang ◽  
Zhang Yanzhen ◽  
Li Yanle ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Mouse Xenograft Model ◽  
Mechanistic Investigation

Abstract Background: Long non-coding RNA (lncRNA) termed small nucleolar RNA host gene 22 (SNHG22) has been reported as a crucial regulator in several types of human cancers. In this study, we aimed to evaluate the function and mechanism of SNHG22 in colorectal cancer (CRC) progression. Methods: Quantitative RT-PCR (qRT-PCR) was used to detect the expression of SNHG22 in adenoma, tumor tissues (TTs), and adjacent nontumorous tissues (ANTs). The biological behaviors of SNHG22 in CRC cell lines were explored both in vitro (CCK-8 assay, flow cytometry, wound scratch, and transwell assays) and in vivo (nude mouse xenograft model). The interaction between SNHG22 and miR-128-3p, and the target genes of miR-128-3p were explored by online tools, qRT-PCR, western blot, and dual-luciferase reporter assay. Results: SNHG22 expression was gradually upregulated in ANTs, adenoma, and TTs. High expression levels of SNHG22 were significantly related to advanced clinicopathological factors and worse survival in patients with CRC. SNHG22 knockdown markedly prohibited CRC cell proliferation, migration, and invasion; and drove cell apoptosis in vitro; and hindered tumor growth in vivo. Mechanistic investigation showed that SNHG22 could bind to microRNA-128-3p (miR-128-3p) and attenuate its inhibitory effects on the expression levels and activity of E2F3. Rescue experiments exhibited that miR-128-3p inhibition or E2F3 upregulation can offset the functions of SNHG22 knockdown in CRC cells. Conclusion: Our findings support the existence of an interactive regulatory network of SNHG22, miR-128-3p, and E2F3 in CRC cell lines, indicating that the SNHG22/miR-128-3p/E2F3 axis is a novel diagnostic and therapeutic target in CRC.

scholarly journals miR-100-3p inhibits cell proliferation and induces apoptosis in human gastric cancer through targeting to BMPR2

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Chun-Wei Peng ◽  
Ling-Xiao Yue ◽  
Yuan-Qin Zhou ◽  
Sai Tang ◽  
Chen Kan ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Cell Lines ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Human Gastric Cancer ◽  
Over Expression ◽  
Qrt Pcr

Abstract Background miR-100 has been reported to closely associate with gastric cancer (GC) initiation and progression. However, the underlying mechanism of miR-100-3p in GC is still largely unclear. In this study, we intend to study how miR-100-3p regulates GC malignancy. Methods The expression levels of miR-100-3p in vitro (GES-1 and GC cell lines) and in vivo (cancerous and normal gastric tissues) were examined by quantitative real-time PCR (qRT-PCR). MTT and PE/Annexin V analyses were responsible for measurement of the effects of miR-100-3p on GC cell proliferation and apoptosis. Transwell assay with or without matrigel was used to examine the capacity of migration and invasion in GC cells. The interaction of miR-100-3p with bone morphogenetic protein receptor 2 (BMPR2) was confirmed through transcriptomics analysis and luciferase reporter assay. qRT-PCR and Western blot analyses were applied to determine the expression of ERK/AKT and Bax/Bcl2/Caspase3, which were responsible for the dysfunction of miR-100-3p. Results miR-100-3p was down-regulated in GC cell lines and cancerous tissues, and was negatively correlated with BMPR2. Loss of miR-100-3p promoted tumor growth and BMPR2 expression. Consistently, the effects of miR-100-3p inhibition on GC cells were partially neutralized by knockdown of BMPR2. Over-expression of miR-100-3p simultaneously inhibited tumor growth and down-regulated BMPR2 expression. Consistently, over-expression of BMPR2 partially neutralized the effects of miR-100-3p over-expression. Further study demonstrated that BMPR2 mediated the effects downstream of miR-100-3p, which might indirectly regulate ERK/AKT and Bax/Bcl2/Caspase3 signaling pathways. Conclusion miR-100-3p acted as a tumor-suppressor miRNA that down-regulated BMPR2, which consequently inhibited the ERK/AKT signaling and activated Bax/Bcl2/Caspase3 signaling. This finding provided novel insights into GC and could contribute to identify a new diagnostic and therapeutic target.

scholarly journals Long Non-coding RNA LINC01969 Promotes Ovarian Cancer by Regulating the miR-144-5p/LARP1 Axis as a Competing Endogenous RNA

2021 ◽  
Vol 8 ◽  
Author(s):  
Jinxin Chen ◽  
Xiaocen Li ◽  
Lu Yang ◽  
Jingru Zhang
Keyword(s):  
Ovarian Cancer ◽  
Rna Binding ◽  
Expression Profiles ◽  
Luciferase Reporter ◽  
Mesenchymal Transition ◽  
Expression Levels ◽  
Lncrna Expression ◽  
Qrt Pcr

Accumulating evidence has shown that long non-coding RNAs (lncRNAs) can be used as biological markers and treatment targets in cancer and play various roles in cancer-related biological processes. However, the lncRNA expression profiles and their roles and action mechanisms in ovarian cancer (OC) are largely unknown. Here, we assessed the lncRNA expression profiles in OC tissues from The Cancer Genome Atlas (TCGA) database, and one upregulated lncRNA, LINC01969, was selected for further study. LINC01969 expression levels in 41 patients were verified using quantitative real-time polymerase chain reaction (qRT-PCR). The in vitro effects of LINC01969 on OC cell migration, invasion, and proliferation were determined by the CCK-8, ethynyl-2-deoxyuridine (EdU), wound healing, and Transwell assays. Epithelial–mesenchymal transition (EMT) was evaluated using qRT-PCR and Western blotting. The molecular mechanisms of LINC01969 in OC were assessed through bioinformatics analysis, RNA-binding protein immunoprecipitation (RIP), dual luciferase reporter gene assays, and a rescue experiment. Finally, in vivo experiments were conducted to evaluate the functions of LINC01969. The results of the current study showed that LINC01969 was dramatically upregulated in OC, and patients with lower LINC01969 expression levels tended to have better overall survival. Further experiments demonstrated that LINC01969 promoted the migration, invasion, and proliferation of OC cells in vitro and sped up tumor growth in vivo. Additionally, LINC01969, which primarily exists in the cytoplasm, boosted LARP1 expression by sponging miR-144-5p and promoted the malignant phenotypes of OC cells. In conclusion, the LINC01969/miR-144-5p/LARP1 axis is a newly identified regulatory signaling pathway involved in OC progression.

scholarly journals Soufeng sanjie formula alleviates collagen-induced arthritis in mice by inhibiting Th17 cell differentiation

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Di Hua ◽  
Jie Yang ◽  
Qinghai Meng ◽  
Yuanyuan Ling ◽  
Qin Wei ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Lymph Nodes ◽  
Inflammatory Cytokines ◽  
Th17 Cell ◽  
Collagen Induced Arthritis ◽  
Expression Levels ◽  
Th17 Cell Differentiation ◽  
Spleen And Lymph Nodes ◽  
Seed Coats

Abstract Background Rheumatoid arthritis (RA) is a chronic autoimmune disease. Soufeng sanjie formula (SF), which is composed of scolopendra (dried body of Scolopendra subspinipes mutilans L. Koch), scorpion (dried body of Buthus martensii Karsch), astragali radix (dried root of Astragalus membranaceus (Fisch.) Bge), and black soybean seed coats (seed coats of Glycine max (L.) Merr), is a traditional Chinese prescription for treating RA. However, the mechanism of SF in treating RA remains unclear. This study was aim to investigate the anti-arthritic effects of SF in a collagen-induced arthritis (CIA) mouse model and explore the mechanism by which SF alleviates arthritis in CIA mice. Methods For in vivo studies, female DBA/1J mice were used to establish the CIA model, and either SF (183 or 550 mg/kg/day) or methotrexate (MTX, 920 mg/kg, twice/week) was orally administered to the mice from the day of arthritis onset. After administration for 30 days, degree of ankle joint destruction and serum levels of IgG and inflammatory cytokines were determined. The balance of Th17/Treg cells in the spleen and lymph nodes was analyzed using flow cytometry. Moreover, the expression levels of retinoic acid receptor-related orphan nuclear receptor (ROR) γt and phosphorylated STAT3 (pSTAT3, Tyr705) in the spleen were detected by immunohistochemistry. Furthermore, the effect of SF on Th17 cells differentiation in vitro was investigated in CD4+ T cells under Th17 polarization conditions. Results SF decreased the arthritis score, ameliorated paw swelling, and reduced cartilage loss in the joint of CIA mice. In addition, SF decreased the levels of bovine collagen-specific IgG in sera of CIA mice. SF decreased the levels of inflammatory cytokines (TNF-α, IL-6, and IL-17A) and increased the level of IL-10 both in the sera and the joint of CIA mice. Moreover, SF treatment rebalanced the Th17/Treg ratio in the spleen and lymph nodes of CIA mice. SF also reduced the expression levels of ROR γt and pSTAT3 (Tyr705) in the spleen of CIA mice. In vitro, SF treatment reduced Th17 cell generation and IL-17A production and inhibited the expression of RORγt, IRF4, IL-17A, and pSTAT3 (Tyr705) under Th17 polarization conditions. Conclusions Our results suggest that SF exhibits anti-arthritic effects and restores Th17/Treg homeostasis in CIA mice by inhibiting Th17 cell differentiation.

scholarly journals METTL14 promotes tumorigenesis by regulating lncRNA OIP5-AS1/miR-98/ADAMTS8 signaling in papillary thyroid cancer

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Xiaoping Zhang ◽  
Dan Li ◽  
Chengyou Jia ◽  
Haidong Cai ◽  
Zhongwei Lv ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Thyroid Cancer ◽  
Papillary Thyroid Cancer ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Papillary Thyroid ◽  
Qrt Pcr ◽  
The Relationship

Abstract Background Papillary thyroid cancer (PTC) is the most common type of cancer of the endocrine system. Long noncoding RNAs (lncRNAs) are emerging as a novel class of gene expression regulators associated with tumorigenesis. Through preexisting databases available for differentially expressed lncRNAs in PTC, we uncovered that lncRNA OIP5-AS1 was significantly upregulated in PTC tissues. However, the function and the underlying mechanism of OIP5-AS1 in PTC are poorly understood. Methods Expression of lncRNA OIP5-AS1 and miR-98 in PTC tissue and cells were measured by quantitative real-time PCR (qRT-PCR). And expression of METTL14 and ADAMTS8 in PTC tissue and cells were measured by qRT-PCR and western blot. The biological functions of METTL14, OIP5-AS1, and ADAMTS8 were examined using MTT, colony formation, transwell, and wound healing assays in PTC cells. The relationship between METTL14 and OIP5-AS1 were evaluated using RNA immunoprecipitation (RIP) and RNA pull down assay. And the relationship between miR-98 and ADAMTS8 were examined by luciferase reporter assay. For in vivo experiments, a xenograft model was used to investigate the effects of OIP5-AS1 and ADAMTS8 in PTC. Results Functional validation revealed that OIP5-AS1 overexpression promotes PTC cell proliferation, migration/invasion in vitro and in vivo, while OIP5-AS1 knockdown shows an opposite effect. Mechanistically, OIP5-AS1 acts as a target of miR-98, which activates ADAMTS8. OIP5-AS1 promotes PTC cell progression through miR-98/ADAMTS8 and EGFR, MEK/ERK pathways. Furthermore, RIP and RNA pull down assays identified OIP5-AS1 as the downstream target of METTL14. Overexpression of METTL14 suppresses PTC cell proliferation and migration/invasion through inhibiting OIP5-AS1 expression and regulating EGFR, MEK/ERK pathways. Conclusions Collectively, our findings demonstrate that OIP5-AS1 is a METTL14-regulated lncRNA that plays an important role in PTC progression and offers new insights into the regulatory mechanisms underlying PTC development.

scholarly journals Downregulation of miR-383 reduces depression-like behavior through targeting Wnt family member 2 (Wnt2) in rats

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shanshan Liu ◽  
Qing Liu ◽  
Yanjie Ju ◽  
Lei Liu
Keyword(s):  
Inflammatory Response ◽  
Family Member ◽  
Chronic Stress ◽  
Hippocampal Neurons ◽  
Luciferase Reporter ◽  
Mild Stress ◽  
Neural Injury ◽  
Expression Levels ◽  
Qrt Pcr

AbstractThis study aimed to evaluate the role of miR-383 in the regulation of Wnt-2 signaling in the rat model of chronic stress. The male SD rats with depressive-like behaviors were stimulated with chronic unpredictable mild stress (CUMS) including ice-water swimming for 5 min, food deprivation for 24 h, water deprivation for 24 h, stimulating tail for 1 min, turning night into day, shaking for 15 min (once/s), and wrap restraint (5 min/time) every day for 21 days. The expression levels of miRNAs were detected by qRT-PCR, and the expression levels of Wnt2, depression-impacted proteins (GFAP, BDNF, CREB), brain neurotransmitters (5-HT, NE, DA) and apoptosis-related proteins (Bax and Bcl-2) were evaluated by qRT-PCR and western blot. Bioinformatic analysis and luciferase reporter assay were performed to determine the relationship between miR-383 and Wnt2. Ethological analysis was evaluated by sugar preference test, refuge island test and open field tests. Rescue experiments including knockdown of miR-383, overexpression and silencing of Wnt2 were performed to determine the role of miR-383. High expression levels of miR-383 were observed in the hippocampus of rats submitted to CUMS model. Downregulation of miR-383 significantly inhibited the apoptosis and inflammatory response of hippocampal neurons, and increased the expression levels of GFAP, BDNF and CREB which were impacted in depression, as well as neurotransmitters, then attenuated neural injury in rats induced by CUMS. Furthermore, Wnt family member 2 (Wnt2) was identified as a target of miR-383, and silencing of Wnt2 obviously attenuated the protective effect of miR-383 inhibitor on the apoptosis and inflammatory response in hippocampal neurons, as well as neural injury in CUMS-induced rats. Downregulation of miR-383 ameliorated the behavioral and neurochemical changes induced by chronic stress in rats by directly targeting Wnt2, indicating that the miR-383/Wnt2 axis might be a potential therapeutic target for MDD.

scholarly journals Babesia bovis-Stimulated Macrophages Express Interleukin-1β, Interleukin-12, Tumor Necrosis Factor Alpha, and Nitric Oxide and Inhibit Parasite Replication In Vitro

2000 ◽  
Vol 68 (9) ◽  
pp. 5139-5145 ◽  
Author(s):  
Lisl K. M. Shoda ◽  
Guy H. Palmer ◽  
Jorge Florin-Christensen ◽  
Monica Florin-Christensen ◽  
Dale L. Godson ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Inflammatory Cytokines ◽  
Tumor Necrosis ◽  
Acute Infection ◽  
Babesia Bovis ◽  
Necrosis Factor Alpha ◽  
Parasite Replication ◽  
Factor Alpha ◽  
Necrosis Factor

ABSTRACT The tick-transmitted hemoparasite Babesia bovis causes an acute infection that results in persistence and immunity against challenge infection in cattle that control the initial parasitemia. Resolution of acute infection with this protozoal pathogen is believed to be dependent on products of activated macrophages (Mφ), including inflammatory cytokines and nitric oxide (NO) and its derivatives.B. bovis stimulates inducible nitric oxide synthase (iNOS) and production of NO in bovine Mφ, and chemical donors of NO inhibit the growth of B. bovis in vitro. However, the induction of inflammatory cytokines in Mφ by babesial parasites has not been described, and the antiparasitic activity of NO produced by B. bovis-stimulated Mφ has not been definitively demonstrated. We report that monocyte-derived Mφ activated by B. bovisexpressed enhanced levels of inflammatory cytokines interleukin-1β (IL-1β), IL-12, and tumor necrosis factor alpha that are important for stimulating innate and acquired immunity against protozoal pathogens. Furthermore, a lipid fraction of B. bovis-infected erythrocytes stimulated iNOS expression and NO production by Mφ. Cocultures of Mφ and B. bovis-infected erythrocytes either in contact or physically separated resulted in reduced parasite viability. However, NO produced by bovine Mφ in response to B. bovis-infected erythrocytes was only partially responsible for parasite growth inhibition, suggesting that additional factors contribute to the inhibition of B. bovis replication. These findings demonstrate that B. bovis induces an innate immune response that is capable of controlling parasite replication and that could potentially result in host survival and parasite persistence.

scholarly journals Polymicrobial Infection with Periodontal Pathogens Specifically Enhances MicroRNA miR-146a in ApoE−/−Mice during Experimental Periodontal Disease

2011 ◽  
Vol 79 (4) ◽  
pp. 1597-1605 ◽  
Author(s):  
Md A. Nahid ◽  
Mercedes Rivera ◽  
Alexandra Lucas ◽  
Edward K. L. Chan ◽  
L. Kesavalu
Keyword(s):  
Periodontal Disease ◽  
Mirna Expression ◽  
Expression Patterns ◽  
Polymicrobial Infection ◽  
Periodontal Pathogens ◽  
Necrosis Factor Alpha ◽  
Expression Levels ◽  
Tnf Α ◽  
Live Bacteria

ABSTRACTPorphyromonas gingivalis,Treponema denticola, andTannerella forsythiaare periodontal pathogens associated with the etiology of adult periodontitis as polymicrobial infections. Recent studies demonstrated that oral infection withP. gingivalisinduces both periodontal disease and atherosclerosis in hyperlipidemic and proatherogenic ApoE−/−mice. In this study, we explored the expression of microRNAs (miRNAs) in maxillas (periodontium) and spleens isolated from ApoE−/−mice infected withP. gingivalis,T. denticola, andT. forsythiaas a polymicrobial infection. miRNA expression levels, including miRNA miR-146a, and associated mRNA expression levels of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) were measured in the maxillas and spleens from mice infected with periodontal pathogens and compared to those in the maxillas and spleens from sham-infected controls. Furthermore, in response to these periodontal pathogens (as mono- and polymicrobial heat-killed and live bacteria), human THP-1 monocytes demonstrated similar miRNA expression patterns, including that of miR-146a,in vitro. Strikingly, miR-146a had a negative correlation with TNF-α secretionin vitro, reducing levels of the adaptor kinases IL-1 receptor-associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF6). Thus, our studies revealed a persistent association of miR-146a expression with these periodontal pathogens, suggesting that miR-146a may directly or indirectly modulate or alter the chronic periodontal pathology induced by these microorganisms.

The Role of Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) in Hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1362-1362
Author(s):  
Yi Shen ◽  
Valerie Barbier ◽  
Ingrid G Winkler ◽  
Jean Hendy ◽  
Jean-Pierre Levesque
Keyword(s):  
Endothelial Cells ◽  
B Cells ◽  
Biologically Active ◽  
Brdu Incorporation ◽  
Tissue Inhibitor ◽  
Over Expression ◽  
Qrt Pcr ◽  
Cell Counts

Abstract Matrix metalloproteinase (MMP) activity is regulated by tissue inhibitor of metalloproteinases (TIMPs). We found that while TIMP-1 and -2 expressions were unaffected, and TIMP-4 was not expressed, TIMP-3 mRNA expression decreased 10-fold within the bone marrow (BM) during G-CSF induced mobilization. In addition, through reverse zymography, the level of biologically active TIMP-3 protein was also shown to decrease during mobilization. Down-regulation of TIMP-3 may contribute to the accumulation of active MMPs within the BM, allowing for the release of hematopoieticstem/progenitor cells (HSPC) from the BM matrix. By qRT-PCR we have shown 10-fold greater TIMP-3 expression in endosteal mRNA compared to central BM mRNA in mouse femur (p=0.008). To assess which bone associated cell populations expressed the majority of TIMP-3, pooled bones were crushed, collagenase treated and FACS sorted. Mesenchymal progenitors (CD45-Lin-Sca1+) expressed the highest level of TIMP-3 followed by endothelial cells (CD45-Lin-CD31+) and mature osteoblasts (CD45-Lin-Sca1-CD51+). Erythroid progenitors (CD45+Ter119+Kit+), megakaryocyte progenitors (CD45+CD41+Kit+) and megakaryocytes (CD45+CD41+Kit−) from BM were also found to express TIMP-3, but at a level at least 10-fold lower than those of non-hematopoietic stromal cells. All other BM hematopoietic cell types tested were negative for TIMP-3 expression. Immunohistofluorescence on bone sections validated TIMP-3 expression in megakaryocytes, endothelial cells and osteoblasts. Expression of TIMP-3 in mouse platelets was confirmed by reverse zymography. To investigate TIMP-3 function we over-expressed huTIMP-3 in mice via retroviral transduction with MND-X-IRES-eGFP (MXIE) retroviral vector. BM cells retrovirally transduced with MXIE-huTIMP-3 or empty MXIE control was transplanted into lethally irradiated congenic mice. Engraftment and transduction levels were determined by GFP expression. At 3-months post-transplant there were no significant differences in body weight, total blood, spleen or BM cell counts between the two groups. qRT-PCR data showed that over-expressing huTIMP-3 did not alter the expression level of endogenous mTIMP-3. Flow cytometry analysis showed that in mice transduced with MXIE-huTIMP-3, the frequency of GFP+ B cells (CD11b-B220+) was reduced by 50% in the blood from 23.88±12.00% to 11.94±7.85% (p=0.0315) and by 64% in the BM from 25.06±13.78% to 9.02±7.67% (p=0.0188) when compared to MXIE controls. Conversely, the frequency of GFP+ huTIMP-3 expressing myeloid cells (CD11b+) was significantly increased in the blood from 55.69±17.13% to77.91±6.31% (p=0.0005), BM from 58.67±16.32% to 77.32±12.02% (p=0.0244) and spleen from 14.07±3.75% to 28.82±6.85% (p=0.0002). Unexpectedly, the frequency of untransduced GFP- myeloid and B cells were similar between the two groups. Although huTIMP-3 over-expression did not significantly alter the number of GFP+ HSPC (Linage-Sca1+Kit+, LSK) per femur (MXIE 0.03±0.03%, MXIE-huTIMP-3 0.01±0.01%, p=0.1139), LSK turnover in huTIMP-3 over-expressing cells was increased in vivo from 4.36±2.83% to 13.31±5.61% (p=0.0159) as determined by BrdU incorporation following 3 days of BrdU administration. Similarly, a trend was also observed in vitro after 12days of culture, LSK sorted from MXIE-huTIMP-3 mice proliferate faster than MXIE controls from 2.55^6cells/ml±1.05 to 9.6^6cells/ml±0.54 (p=0.1). In summary, huTIMP-3 over-expression in mice increased HSPC proliferation in vivo and in vitro. And whilst the huTIMP-3 over-expression in mice was not at a sufficient level to observe a global effect on total BM haematopoiesis, our data suggests that forced huTIMP-3 over-expression in vivo skews differentiation towards myelopoiesis at the detriment of lymphopoiesis.

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