A time series study on BMP6 induced osteoblast differentiation of human mesenchymal stem cells (hMSC)
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ABSTRACT: BMP6 mediated osteoblast differentiation plays a key role in skeletal development and bone disease. Unfortunately, the signaling pathways regulated by BMP6 are largely uncharacterized due to both a lack of data and the complexity of the response. To better characterize the signaling pathways responsive to BMP6, we conducted a time series microarray study to track BMP6 induced osteoblast differentiation and mineralization. Human MSC cells underwent osteogenic induction with BMP6 treatment for 0 hours, 8 hours, 24 hours, and 96 hours, which correspond to four phenotypic groups, i.e. control, preosteoblast (no mineralization), (sub-maximal) mineralization, and maximal mineralization at 14 days after the initiation of BMP treatment (18 days in total). Cells were harvested at 8 hours, 24 hours, 96 hours and 10 days for microarray profiling. Assays were run in duplicate for a total of 26 arrays. We only used 20 arrays in the reference paper.
Project description:We have established that BMP6 is an important endogenous regulator of human osteoblast differentiation. Our preliminary experiment showed that 8 hour BMP6 treatment induced early osteoblast markers in hMSC. In this study, we used microarrays to profile the global gene expression program in hMSC induced by BMP6 treatment and further identify the early osteogenic responses to BMP6 stimulation. Experiment Overall Design: The dataset contains a total of 4 gene chip measurements from duplicate experiments each with paired measurements of human MSC with or without 8 hours BMP6 treatment.
Project description:BMP6 mediated osteoblast differentiation plays a key role in skeletal development and bone disease. Unfortunately, the signaling pathways regulated by BMP6 are largely uncharacterized due to both a lack of data and the complexity of the response. To better characterize the signaling pathways responsive to BMP6, we conducted a time series microarray study to track BMP6 induced osteoblast differentiation and mineralization.
Project description:We have established that BMP6 is an important endogenous regulator of human osteoblast differentiation. Our preliminary experiment showed that 8 hour BMP6 treatment induced early osteoblast markers in hMSC. In this study, we used microarrays to profile the global gene expression program in hMSC induced by BMP6 treatment and further identify the early osteogenic responses to BMP6 stimulation. Keywords: Stress response
Project description:Bone morphogenetic proteins (BMP) are firmly implicated as intra-ovarian regulators of follicle development and steroidogenesis. We carried out a microarray analysis to examine global changes in gene expression in bovine theca cells in response to treatment with BMP6 alone and in combination with LH. There was a major effect of BMP6 treatment on the gene expression profile with a much weaker effect of LH. None of these differences in response to LH treatment was found to be statistically significant after applying Benjamini-Hochberg correction. BMP6 significantly (>2-fold; P<0.01) up- or down-regulated expression of 445 genes. Insulin-like peptide 3 (INSL3) was the most heavily down-regulated gene (-43-fold) with CYP17A1 and other key transcripts involved in TC steroidogenesis including LHCGR, INHA, STAR, CYP11A1 and HSD3B1 also down-regulated. BMP6 also reduced expression of NR5A1 encoding steroidogenic factor-1 known to target the promoter regions of the aforementioned genes. Real-time PCR confirmed these findings and also revealed a marked reduction in expression of INSL3 receptor (RXFP2). Secretion of INSL3 protein and androstenedione were also suppressed suggesting a functional link between BMP and INSL3 pathways in controlling androgen synthesis. Theca interna cells (TC) were isolated from adult bovine ovaries obtained from the slaughterhouse. TC pooled from approximately 50 individual 4-6mm follicles were plated out in in 24-well plates (0.5 x106 viable cells/ml/well) and cultured for 6 days under defined serum-free conditions with treatments present on days 3-6 inclusive. TC were treated for 4 days with BMP6 (10 ng/ml) under both basal and LH-stimulated conditions (M-BM-1 160 pg LH/ml). These dose-levels of BMP6 and LH were chosen because they elicited optimal responses in our previous studies. The experiment was replicated four times with TC harvested from different batches of ovaries. Total RNA was isolated using the Ribopure RNA isolation kit (Ambion) according to the manufacturerM-bM-^@M-^Ys instructions. RNA yield and quality were evaluated by spectrophotometry at 260/280 nm and agarose gel electrophoresis before submitting 5 M-NM-<g of each RNA sample (n = 16 comprising 4 biological replicates x 4 treatment conditions) to an accredited Affymetrix service provider for microarray analysis using the bovine genome array GeneChip (n=16 arrays).
Project description:Human pulmonary arterial endothelial cells (PAECs) and blood outgrowth endothelial cells (BOECs) from healthy subjects were stimulated with BMP9, BMP2 or BMP6. and assessed for changes in gene transcription by microarray. Unlike BMP2 and BMP6, which had negligible impacts on gene expression, BMP9 induced the differential regulation of 1883 genes (adjusted P value < 0.05), including several key components of canonical BMP signaling such as ID1, ID2 and BMPR2.
Project description:Hepcidin, a peptide hormone that decreases intestinal iron absorption and macrophage iron release, is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. Endogenous stimulants of Hepcidin transcription include bone morphogenic protein 6 (BMP) and interleukin-6 (IL-6) via effects on Smad4 or Stat3, respectively. We conducted a small-scale chemical screen in zebrafish embryos to identify small molecules that modulate hepcidin expression. We found that treatment with the isoflavone genistein from 28−52 hours post-fertilization in zebrafish embryos enhanced Hepcidin transcript levels as assessed by whole mount in situ hybridization and quantitative realtime RT-PCR. Genistein’s stimulatory effect was conserved in human hepatocytes: genistein treatment of HepG2 cells increased both Hepcidin transcript levels and Hepcidin promoter activity. We found that genistein’s effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either the BMP response elements or the Stat3 binding site in the Hepcidin promoter. RNA-sequencing of transcripts from genistein-treated hepatocytes indicated that genistein upregulated 68% of the transcripts that were upregulated by BMP6, however genistein raised the levels of several transcripts involved in Stat3 signaling that were not upregulated by BMP6. Chromatin-immunoprecipitation and ELISA experiments revealed that genistein enhanced Stat3 binding to the Hepcidin promoter and increased phosphorylation of Stat3 in HepG2 cells. CONCLUSION: Genistein is the first small molecule experimental drug that stimulates Hepcidin expression in vivo and in vitro. These experiments demonstrate the feasibility of identifying and characterizing small molecules that increase Hepcidin expression. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes. RNA-seq of HepG2 cells treated with DMSO 1%, BMP6 50 ng/ml, or genistein 10 micromolar. The numbers of biological replicates were 3, 2, and 3.
Project description:We report the application of next generation sequencing technology for high-throughput profiling of mRNA, circRNA and lncRNA in hFOB 1.19 cells during the mineralization. Osteoblast RNA profiles in different process of mineralization, which is day 0, day 3, day 6 and day 9, respectively. The transcriptome sequencing on an Illumina HiSeq X Ten platform were carried out at Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. In the process of osteoblast mineralization, a total of 8464 dysregulated circRNAs were identified, of which 6332 circRNAs were upregulated and 2132 circRNAs were downregulated. In addition, 1633 differentially expressed mRNAs were also identified, with 1027 mRNAs upregulated and 606 mRNAs downregulated. This study provides a framework for the application of NGS profiling towards characterization of osteoblast mineraliztion.
Project description:Differentiation of human skeletal stem cells (hMSC) into osteoblasts is regulated by a few well described transcription factors. Our study used clustering and gene expression data to identify a novel transcription factor. ZNF25, which we showed is involved in osteoblast differentiation. We used microarrays to study gene expression of hMSC-TERT4 cells during osteoblast differentiation.