Time series gene expression profiling and temporal regulatory pathway analysis of BMP6 induced osteoblast differentiation and mineralization

Background: 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. Results: To better characterize the signaling pathways responsive to BMP6, we conducted a time series microarray study to track BMP6 induced osteoblast differentiation and mineralization. These temporal data were analyzed using a customized gene set analysis approach to identify temporally coherent sets of genes that act downstream of BMP6. Our analysis identified BMP6 regulation of previously reported pathways, such as the TGF-beta pathway. We also identified previously unknown connections between BMP6 and pathways such as Notch signaling and the MYB and BAF57 regulatory modules. In addition, we identify a super-network of pathways that are sequentially activated following BMP6 induction. Conclusion: In this work, we carried out a microarray-based temporal regulatory pathway analysis of BMP6 induced osteoblast differentiation and mineralization using GAGE method. This novel temporal analysis is more informative and powerful than the classical static pathway analysis in that: (1) it captures the interconnections between signaling pathways or functional modules and demonstrates the even higher level organization of molecular biological systems; (2) it describes the temporal perturbation patterns of each pathway or module and their dynamic roles in osteoblast differentiation. The same set of experimental and computational strategies employed in our work could be useful for studying other complex biological processes

Gene Regulatory Network Reconstruction and Pathway Inference from High Throughput Gene Expression Data.

Two basic motivating questions in biomedical research are: What genes regulate what other genes? What genes or groups of genes regulate a specific phenotype? Gene regulatory network (GRN) reconstruction and pathway inference are the two computational strategies addressing these two questions respectively. GRN reconstruction is to infer the components and topology of an unknown pathway, while pathway inference is to infer association between known pathways and a phenotype. This thesis focuses on gene regulatory network reconstruction and pathway inference from high throughput biological data. In the first part of this work, I developed a novel method, MI3, for de novo GRN reconstruction using continuous three-way mutual information. MI3 addresses three major issues in previous probabilistic methods simultaneously: (1) to handle continuous variables, (2) to detect high order relationships, (3) to differentiate causal vs. confounding relationships. MI3 consistently and significantly outperformed frequently used control methods and faithfully capture mechanistic relationships from gene expression data. In the second part of this work, I proposed another novel method, GAGE, Generally Applicable Gene Set Enrichment for pathway inference. I successfully apply GAGE to multiple microarray data sets with different sample sizes, experimental designs and profiling techniques. GAGE shows significantly better performance when compared to two other commonly used GSA methods of GSEA and PAGE. GAGE reveals novel and relevant regulatory mechanisms from both published and previously unpublished microarray studies. In the third part of this work, we conducted a microarray study on transcriptional programs during BMP6 induced osteoblast differentiation and mineralization, and applied GAGE to recover the regulatory pathways and transcriptional signaling networks in the process. I not only showed which pathways or gene sets are significant, but also when and how they are involved in the osteoblast differentiation and mineralization. Different from common pathway analyses, our work further captures the interconnections among individual pathways or functional groups and integrate them into a whole system.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/61768/1/luow_1.pd

Canonical Notch signaling is required for bone morphogenetic protein‐mediated human osteoblast differentiation

Osteoblast differentiation of bone marrow‐derived human mesenchymal stem cells (hMSC) can be induced by stimulation with canonical Notch ligand, Jagged1, or bone morphogenetic proteins (BMPs). However, it remains elusive how these two pathways lead to the same phenotypic outcome. Since Runx2 is regarded as a master regulator of osteoblastic differentiation, we targeted Runx2 with siRNA in hMSC. This abrogated both Jagged1 and BMP2 mediated osteoblastic differentiation, confirming the fundamental role for Runx2. However, while BMP stimulation increased Runx2 and downstream Osterix protein expression, Jagged1 treatment failed to upregulate either, suggesting that canonical Notch signals require basal Runx2 expression. To fully understand the transcriptomic profile of differentiating osteoblasts, RNA sequencing was performed in cells stimulated with BMP2 or Jagged1. There was common upregulation of ALPL and extracellular matrix genes, such as ACAN, HAS3, MCAM, and OLFML2B. Intriguingly, genes encoding components of Notch signaling (JAG1, HEY2, and HES4) were among the top 10 genes upregulated by both stimuli. Indeed, ALPL expression occurred concurrently with Notch activation and inhibiting Notch activity for up to 24 hours after BMP administration with DAPT (a gamma secretase inhibitor) completely abrogated hMSC osteoblastogenesis. Concordantly, RBPJ (recombination signal binding protein for immunoglobulin kappa J region, a critical downstream modulator of Notch signals) binding could be demonstrated within the ALPL and SP7 promoters. As such, siRNA‐mediated ablation of RBPJ decreased BMP‐mediated osteoblastogenesis. Finally, systemic Notch inhibition using diabenzazepine (DBZ) reduced BMP2‐induced calvarial bone healing in mice supporting the critical regulatory role of Notch signaling in BMP‐induced osteoblastogenesis.Bone morphogenetic protein (BMP) stimulation of bone‐marrow‐derived human mesenchymal progenitor cells (hMSCs) increases Notch proteins via increased Notch ligand Jagged1 expression. Canonical Notch signaling is required for BMP‐induced ALPL expression and osteoblastic commitment of hMSCs. Both BMP‐induced osteoblastogenesis and Notch‐induced osteoblastogenesis require Runx2.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162778/2/stem3245_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162778/1/stem3245.pd

Kinetics of gene expression and bone remodelling in the clinical phase of collagen-induced arthritis

INTRODUCTION: Pathological bone changes differ considerably between inflammatory arthritic diseases and most studies have focused on bone erosion. Collagen-induced arthritis (CIA) is a model for rheumatoid arthritis, which, in addition to bone erosion, demonstrates bone formation at the time of clinical manifestations. The objective of this study was to use this model to characterise the histological and molecular changes in bone remodelling, and relate these to the clinical disease development. METHODS: A histological and gene expression profiling time-course study on bone remodelling in CIA was linked to onset of clinical symptoms. Global gene expression was studied with a gene chip array system. RESULTS: The main histopathological changes in bone structure and inflammation occurred during the first two weeks following the onset of clinical symptoms in the joint. Hereafter, the inflammation declined and remodelling of formed bone dominated. Global gene expression profiling showed simultaneous upregulation of genes related to bone changes and inflammation in week 0 to 2 after onset of clinical disease. Furthermore, we observed time-dependent expression of genes involved in early and late osteoblast differentiation and function, which mirrored the histopathological bone changes. The differentially expressed genes belong to the bone morphogenetic pathway (BMP) and, in addition, include the osteoblast markers integrin-binding sialoprotein (Ibsp), bone gamma-carboxyglutamate protein (Bglap1), and secreted phosphoprotein 1 (Spp1). Pregnancy-associated protein A (Pappa) and periostin (Postn), differentially expressed in the early disease phase, are proposed to participate in bone formation, and we suggest that they play a role in early bone formation in the CIA model. Comparison to human genome-wide association studies (GWAS) revealed differential expression of several genes associated with human arthritis. CONCLUSIONS: In the CIA model, bone formation in the joint starts shortly after onset of clinical symptoms, which results in bony fusion within one to two weeks. This makes it a candidate model for investigating the relationship between inflammation and bone formation in inflammatory arthritis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13075-015-0531-7) contains supplementary material, which is available to authorized users

Alendronate Alters Single Cell Gene Expression of Cortical Osteoblast Lineage Cells During Bone Loss

The mineralized matrix of bone makes it difficult to examine specific populations of cells which are integral to the tissue using traditional molecular methods. For this study we examined single cell cortical osteoblasts derived from the femurs of C57BL/6J mice. After isolating single cells from bone, we were able to individually analyze their gene expression profiles using the quantitative polymerase chain reaction (qPCR). The mice used for the study were divided into 4 treatment groups, including ovariectomized mice (OVX) and sham surgical controls (SHAM), treated with or without the anti-resorptive bisphosphonate drug Alendronate, an effective FDA approved therapeutic for slowing bone loss in association with osteoporosis. To administer Alendronate, mice were treated with 100 μg/kg Alendronate weekly via intraperitoneal (IP) injections. After a 16- week period post-surgery, mice were euthanized and bone tissue, including spines and femurs, were preserved for analysis (n= 10 mice per treatment per time point). Single cell cortical osteoblasts were obtained from preserved femur through serial collagenase digestion. Osteoblasts cells were collected by selecting for CD31-/CD45-/Alkaline Phosphatase+ using fluorescence-activated cell sorting (FACS). Over 100 osteoblast cells per treatment group were analyzed using qPCR for the detection of 96 specific transcripts. We confirmed the effects of bone loss in the mice due to OVX surgery via CT scans of the collected spines and femurs at a resolution of 9μm. We identified unique expression patterns in each treatment subset of osteoblast cells. The single cell gene expression analysis revealed that the cell populations that were undergoing bone loss had genetic signatures with distinctive gene expression profiles. Analysis of cells at a single cell level may lead to a better understanding of the effects of anti-resorptive agents on the cell populations found within bone

Effect of NF-κB Haploinsufficiency on Calvarial Bone Healing

Bone defect is a major and challenging health concern. The treatment for bone defect aims to enhance bone regeneration, which is highly regulated by many molecular signaling pathways. Growing evidence suggested that proper inflammatory signaling was crucial for bone regeneration. Previous study showed that treatment of MSCs with expression of NF-κB increased MSCs engraftment in damaged tissue. Previous work in our lab indicated a role of NF-κB on osteoblast differentiation during physiological bone development. The present study was designed to study role of NF-κB signaling in bone healing using genetically-modified mouse with haploinsufficiency of p65 in osteoblasts. Here, we showed that mice with osteoprogenitor-specific NF-κB haploinsufficiency displayed reduced calvarial defect bone repair manifested by micro-CT and histological analysis. The progenitor cells from p65 haploinsufficient mice demonstrated fewer CFU-OB colonies and decreased osteoblastic markers expression (Sp7, Alp and Bsp) in response to rhBMP2. Furthermore, rhBMP2 mediated Smad phosphorylation was disrupted in the absence of sufficient p65 signal. Therefore, we concluded that NF-κB haploinsufficiency impairs bone repair by downregulation of BMP2 mediated canonical Smads signaling and osteogenic differentiation. The effect of inflammatory mediators on bone formation was complex and yet to be elucidated. Based on our findings, we proposed a direct regulatory role of NF-κB in rhBMP2 mediated bone repair, suggesting that sufficient inflammatory cues are essential for bone regeneration. Uncovering the function of NF-κB in MSC-mediated repair will improve understanding of bone regeneration mechanism and provide a clue for bone regenerative therapy for treatment of bony defect.Doctor of Philosoph

Cellular biology of fracture healing

The biology of bone healing is a rapidly developing science. Advances in transgenic and gene‐targeted mice have enabled tissue and cell‐specific investigations of skeletal regeneration. As an example, only recently has it been recognized that chondrocytes convert to osteoblasts during healing bone, and only several years prior, seminal publications reported definitively that the primary tissues contributing bone forming cells during regeneration were the periosteum and endosteum. While genetically modified animals offer incredible insights into the temporal and spatial importance of various gene products, the complexity and rapidity of healing—coupled with the heterogeneity of animal models—renders studies of regenerative biology challenging. Herein, cells that play a key role in bone healing will be reviewed and extracellular mediators regulating their behavior discussed. We will focus on recent studies that explore novel roles of inflammation in bone healing, and the origins and fates of various cells in the fracture environment. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop ResAdvances in transgenic and gene‐targeted mice have enabled tissue and cell‐specific investigation of skeletal regeneration. While genetically modified animals offer incredible insights into the temporal and spatial importance of various molecules, the complexity and rapidity of healing renders studies of regenerative biology challenging. Herein, cells and extracellular mediators that play a key role in bone healing are reviewed. We will focus on recent studies that explore the origins and fates of various cells in the fracture environment.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/148261/1/jor24170_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148261/2/jor24170-sup-0002-SuppTab-S2.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148261/3/jor24170-sup-0001-SuppTab-S1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148261/4/jor24170.pd

Genes and molecular pathways of the osteogenic process

This chapter will provide an up-to-date depiction of the molecular networks involved in the osteogenic process, focusing on main genes and signaling pathways (schematically represented in Figure 1), whose integrity is required for the correct skeletal morphogenesis and patterning and for maintaining bone homeostasis. Particular attention will be devoted to list and dissect the human syndromes and disorders associated to genes belonging to the main osteogenic pathways, with regard to the skeletal phenotyp

Building Bone: Human mesenchymal stromal cells and the identification of genes and processes in osteoblast differentiation

This thesis presents a number of novel findings in the bone biology field using a combination of bioinformatic, genomic, molecular, and proteomic approaches, and highlights the complexity of the biology and study of osteoblast differentiation. The results described within include the discovery of number of fa