ABSTRACT: Osteoblasts represent an important cell type playing a role in not only bone formation but also regulate hematopoiesis by secreting factor as well as via contact with hematopoietic stem cells in the bone marrow. Since Wnt signalling plays an important role in osteoblast differentiation, we were interested in looking at how canonical Wnt signalling activation in osteoblastic cells is likely to affect hematopoietic cell adhesion and regulate their fate. Endogenous Wnt signaling activation in osteoblastic SaOS2 cells was achieved by generating doxycycline (DOX) inducible antisense-APC expressing SaOS2 cells. Gene expression profiling was performed on SaOS2 cells induced with DOX (1μg/ml) for 3 days and further on DOX treated cells allowed to recover for 3 days. The results reveal changes in expression of a number of cell adhesion and extracellular matrix protein genes as well as genes involved in osteoblast differentiation. Doxycycline inducible antisense APC expressing SaOS2 cells were treated with DOX for 3 days and compared with control (untreated) cells. In addition, a set of DOX induced cells was further cultured for 3 days in absence of Dox to allow for the cells to recover and see the change in gene expression compared to Dox treated and control cells. All experiments were done in triplicates (in total 9 samples).
Project description:Therapeutic targeting of the Wnt pathway is of high clinical interest for treating bone loss disorders such as osteoporosis. These therapies inhibit the action of negative regulators of osteoblastic Wnt signaling. The observation that Wnt inhibitory factor 1 (WNT1) was epigenetic silencing in osteosarcoma (OS) raised concerns for such a treatment approach. In this study, genome-wide methylation profiling of OS derived from mouse models demonstrated Wif1 silencing in OS is not driven by DNA methylation. Treatment of mouse and human OS cells with methylation and HDAC inhibitors showed Wif1 was unresponsive to methylation inhibition but responded robustly to HDAC inhibition. Consistent with an HDAC dependent mechanism of silencing, the Wif1 locus in OS was characterized by low levels of acetylation and a bivalent H3K4/H3K27 trimethylation state. Wif1 expression marked late stages of normal osteoblast development and stratified OS tumours based on differentiation stage across species. Culture of human and mouse OS cells under differentiation inductive conditions increased expression of Wif1 in parallel with known osteoblast differentiation markers. Together these results demonstrate that Wif1 is not targeted for silencing by DNA methylation in OS. The reduced expression of Wif1 in OS cells is in context with their stage in differentiation. 3 cell lines derived from primary tumors from p53 Rb Osterix-Cre:lox OS model, 3 Osteoblasts (differentiated Kusa4b10 cells (21 days under osteoblastic differentiation conditions)
Project description:Cells respond heterogeneously to DNA damage. We engineered genetic circuits to detect differential responses in a population that persist for many days post-stimulus. We used microarrays to compare memory and non-memory subpopulations 3 days after DNA damage or doxycycline exposure. MD12/p53R2-RE and MD10/TetOx2 cells were either exposed to UV (10uJ/m^2) or doxycycline (1 ug/mL, 24 hours) and allowed to recover 3 days before sortng of memory and non-memory cells and RNA extraction. Two replicates were submitted for each condition (UV memory, UV non-memory, dox memory, dox non-memory)
Project description:Transcription initiation in eukaryotes by RNA polymerase II requires numerous general and regulatory factors including the general transcription factors. Here, we report a new cofactor of the general transcription factor IIF, GAS41, which was previously implicated in tumor development. Microarray analysis of cells with induced overexpression of cofactor GAS41 identifies a significant number of up-regulated transcripts (p-value of 0.01). Keywords: altered gene expression Overall design: Saos2 Tet-Off clone B3-Dox, with induced GAS41 overexpression, was compared to Saos2 Tet-Off clone B3+Dox, without overexpression. Additionally, B3-Dox was compared to Saos2 Tet-Off clones F7 +Dox/ -Dox, both transfected with pTRE2pur response plasmid only, to exclude any doxycycline effect. All experiments were done in triplicates, revealing twelve data sets.
Project description:Primary osteoblast NEMCO cells have been transduced with lentivirus conditionally (doxycycline) expressing RUNX2 and treated with E2 to demonstrate effects of estrogen signaling on RUNX2 response NEMCO cells kept at charcoal stripped serum have been treated with dox and/or E2 for 48h
Project description:The aim of the study was to identify gene expression profiles that distinguished different stages of osteoblast commitment, and determine whether the same osteoblast cell populations from mice of different ages had distinguishable expression profiles that may indicate functional changes are apparent for the cell populations during different stages of skeletal development. Pre-osteoblastic and mature osteoblastic cells were isolated from collagenase digested bones from 5 week old and 8 week wild type C57BL6 mice by FACS based on Lin, Sca-1, CD51, and CD31 cell surface markers. Overall design: Pre-osteoblastic and mature osteoblastic cells were isolated from collagenase digested bones from 5 week old and 8 week wild type C57BL6 mice by FACS based on Lin, Sca-1, CD51, and CD31 cell surface markers.
Project description:The miRNAs play important roles in regulating gene expression at post-transcriptional level through fine-tuning of protein-encoding gene expression, and are involved in regulating important biological processes in different cells and tissue types, including osteoblast differentiation. Canonical Wnt signaling is also known to play pleiotropic roles in regulating cell differentiation during mammalian osteogenesis, although the role of miRNAs is not established for Wnt signaling in osteoblast differentiation. Here we examined the role of candidate miRNAs expressed differentially after Wnt3a expression during osteoblast differentiation. Overexpression of the Wnt3a gene resulted in increased transcription of the ALP gene, but in decreased transcription of the Runx2, Col1a and OCN genes in osteoblastic MC3T3-E1 cells. Analysis of miRNA expression profile showed that 14 miRNAs were up-regulated and 21 miRNAs were down-regulated after Wnt3a overexpression in MC3T3-E1 cells. TGF-beta3 is presumed to be a candidate target gene of one of the down-regulated miRNAs with database search. Transfection of the miRNA mimic into MC3T3-E1 cells significantly inhibited the TGF-beta3 mRNA level and resultant protein expression, although overexpression of the Wnt3a gene could reverse the effect of the miRNA, resulting in the increased TGF-beta3 mRNA and protein levels. These results suggest that the miRNA is involved in osteoblast differentiation as a critical regulatory factor, mediating crosstalk between Wnt3a signaling pathway and TGF-beta3 signaling pathway. Overall design: 4 samples
Project description:Wnt signaling is a major regulator of osteoblast differentiation and function. To investigate how Wnt3a signaling regulates osteoblastic gene expression and to identify the role of Lrp5 and Lrp6 in mediating Wnt3a signaling in osteoblasts, neonatal calvarial osteoblasts isolated from C57Bl6 (WT) and osteoblasts lacking Lrp5 (Lrp5KO), Lrp6 (Lrp6KO) and, both Lrp5 and 6 (Lrp5/6KO) were treated with Wnt3a for 24 hours and gene expression changes were quantified by RNA-seq. Overall design: Transcriptome analysis to identify Wnt3a regulated genes in Wild Type, Lrp5KO , Lrp6KO and Lrp5/6KO osteoblasts
Project description:Extracellular nucleotides are potent signaling molecules mediating cell-specific biological functions. We previously demonstrated that adenosine 5'-triphosphate (ATP) inhibits the proliferation while stimulating the migration, in vitro and in vivo, of human bone marrow-derived mesenchymal stem cells (BM-hMSC). Here, we investigated the effects of ATP on BM-hMSC differentiation capacity. Molecular analysis showed that ATP treatment modulated the expression of several genes (e.g. wnt-pathway-related genes) governing osteoblastic and adipogenic differentiation of MSCs. Functional studies demonstrated that ATP, under specific culture conditions, stimulated adipogenic and osteogenic differentiation by significantly increasing the lipid accumulation and the expression levels of the adipogenic master gene PPARγ (peroxisome proliferator activated receptor-gamma) and by promoting the mineralization and the expression of the osteoblast-related gene RUNX2 (Runt-related transcription factor 2), respectively. BM-hMSCs cells were transiently exposed to ATP 1mM for 24 hours (ATP pre-treatment) before starting differentiation induction. Then, BM-hMSCs were cultured under adipogenic/osteogenic conditions. Gene Expression Profile was performed on differentiate cells after 3 weeks of induction culture.
Project description:VCaP cells expressing inducible shRNAs for either ERG or a non-targeting control were treated with Doxycycline for 1, 3, 7 and 10 days prior to collection This experiment is designed to see which genes and pathways are modulated by ERG knockdown VCaP cells stably expressing a Doxycycline (dox)-inducible control nontargeting shRNA (Pak4) or an ERG shRNA (2217) were exposed to 100ng/ml Dox for the noted days.