75 research outputs found
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
GAGE: generally applicable gene set enrichment for pathway analysis
<p>Abstract</p> <p>Background</p> <p>Gene set analysis (GSA) is a widely used strategy for gene expression data analysis based on pathway knowledge. GSA focuses on sets of related genes and has established major advantages over individual gene analyses, including greater robustness, sensitivity and biological relevance. However, previous GSA methods have limited usage as they cannot handle datasets of different sample sizes or experimental designs.</p> <p>Results</p> <p>To address these limitations, we present a new GSA method called Generally Applicable Gene-set Enrichment (GAGE). We successfully apply GAGE to multiple microarray datasets with different sample sizes, experimental designs and profiling techniques. GAGE shows significantly better results when compared to two other commonly used GSA methods of GSEA and PAGE. We demonstrate this improvement in the following three aspects: (1) consistency across repeated studies/experiments; (2) sensitivity and specificity; (3) biological relevance of the regulatory mechanisms inferred.</p> <p>GAGE reveals novel and relevant regulatory mechanisms from both published and previously unpublished microarray studies. From two published lung cancer data sets, GAGE derived a more cohesive and predictive mechanistic scheme underlying lung cancer progress and metastasis. For a previously unpublished BMP6 study, GAGE predicted novel regulatory mechanisms for BMP6 induced osteoblast differentiation, including the canonical BMP-TGF beta signaling, JAK-STAT signaling, Wnt signaling, and estrogen signaling pathwaysāall of which are supported by the experimental literature.</p> <p>Conclusion</p> <p>GAGE is generally applicable to gene expression datasets with different sample sizes and experimental designs. GAGE consistently outperformed two most frequently used GSA methods and inferred statistically and biologically more relevant regulatory pathways. The GAGE method is implemented in R in the "gage" package, available under the GNU GPL from <url>http://sysbio.engin.umich.edu/~luow/downloads.php</url>.</p
OsteoblastāTargeted suppression of PPARĪ³ increases osteogenesis through activation of mTOR signaling
Nuclear receptor peroxisome proliferatorāactivated receptorāĪ³ (PPARĪ³) is an essential transcription factor for adipocyte differentiation. In mesenchymal stem cells, PPARĪ³ has been assumed to play a negative role in osteoblastic differentiation, by working in an adipogenesis dependent manner, due to the reciprocal relationship between osteoblast and adipocyte differentiation. However, the direct role of PPARĪ³ in osteoblast function is not fully understood, due in part to inadequate model systems. Here, we describe an adenoviralāmediated PPARĪ³ knockout system in which suppression of PPARĪ³ in mesenchymal stem cells enhanced osteoblast differentiation and inhibited adipogenesis in vitro. Consistent with this in vitro observation, lipoatrophic AāZIP/F1 mice, which do not form adipocytes, displayed a phenotype in which both cortical and trabecular bone was significantly increased compared with wildātype mice. We next developed an inducible osteoblastātargeted PPARĪ³ knockout ( Osx Cre/floxā PPARĪ³ ) mouse to determine the direct role of PPARĪ³ in bone formation. Data from both in vitro cultures of mesenchymal stem cells and in vivo ĀµCT analysis of bones suggest that suppression of PPARĪ³ activity in osteoblasts significantly increased osteoblast differentiation and trabecular number. Endogenous PPARĪ³ in mesenchymal stem cells and osteoblasts strongly inhibited Akt/mammalian target of rapamycin (mTOR)/p70S6k activity and led to decreased osteoblastic differentiation. Therefore, we conclude that PPARĪ³ modulates osteoblast differentiation and bone formation through both direct and indirect mechanisms. The direct mode, as shown here, involves PPARĪ³ regulation of the mTOR pathway, while the indirect pathway is dependent on the regulation of adipogenesis. S tem C ells 2013;31:2183ā2192Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/100308/1/stem1455.pd
Leptin Functions Peripherally to Regulate Differentiation of Mesenchymal Progenitor Cells
Leptin functions through a well-documented central neuroendocrine pathway to regulate bone mass. However, the ability of leptin to modulate bone mass through a peripheral mechanism has been debated due to conflicting in vitro results and lack of sufficient in vivo models. We utilized mice with LoxP sites introduced into the long-form leptin receptor (ObRb) gene to determine how leptin regulates mesenchymal progenitor cell (MPC) differentiation and osteoblast function in vitro and in vivo . Rapid phosphorylation of Stat3 after leptin treatment of bone marrow stromal cells (BMSCs) from mice with conditional deletion of ObRb in macrophages (LysM Cre+F/F ) confirmed expression of functional leptin receptors by BMSCs. Adenovirus-Cre mediated disruption of ObRb in primary stromal cells decreased mineralization and increased adipogenesis. In contrast, BMSCs harvested from leptin-signaling deficient Ob/Ob or Db/Db mice showed increased mineralization. To determine the physiologic relevance of these differences, mice with cell-specific deletion of ObRb in mesenchymal precursors (3.6 Cre+F/F ) or osteoblasts (2.3 Cre+F/F ) were generated. Although the 2.3 Cre+F/F mice were grossly normal, the 3.6 Cre+F/F mice displayed mild obesity that was not attributed to food intake. Femurs of 3.6 Cre+F/F animals showed a 58%ā61.9% increase in trabecular bone volume and a 65.5%ā74% increase in bone mineral density. Cortical volume and mineral content were also increased 18%ā22%. Primary 3.6 Cre+F/F BMSCs recapitulated the high mineralization phenotype of Ob/Ob and Db/Db BMSCs. We conclude that leptin may have multiple peripheral roles depending on the differentiation state of MPC. Leptin (a) helps maintain MPCs in an undifferentiated state and (b) promotes mineralization of more differentiated osteoblasts. S TEM C ells 2010;28:1071ā1080Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/77453/1/432_ftp.pd
Bone healing in an aged murine fracture model is characterized by sustained callus inflammation and decreased cell proliferation
Geriatric fractures take longer to heal and heal with more complications than those of younger patients; however, the mechanistic basis for this difference in healing is not well understood. To improve this understanding, we investigated cell and molecular differences in fracture healing between 5āmonthāold (young adult) and 25āmonthāold (geriatric) mice healing utilizing highāthroughput analysis of gene expression. Mice underwent bilateral tibial fractures and fracture calluses were harvested at 5, 10, and 20 days postāfracture (DPF) for analysis. Global gene expression analysis was performed using Affymetrix MoGene 1.0 ST microarrays. After normalization, data were compared using ANOVA and evaluated using Principal Component Analysis (PCA), CTen, heatmap, and Incromaps analysis. PCA and crossāsectional heatmap analysis demonstrated that DPF followed by age had pronounced effects on changes in gene expression. Both unāfractured and 20 DPF aged mice showed increased expression of immuneāassociated genes (CXCL8, CCL8, and CCL5) and at 10 DPF, aged mice showed increased expression of matrixāassociated genes, (Matn1, Ucma, Scube1, Col9a1, and Col9a3). Cten analysis suggested an enrichment of CD8+ cells and macrophages in old mice relative to young adult mice and, conversely, a greater prevalence of mast cells in young adult mice relative to old. Finally, consistent with the PCA data, the classic bone healing pathways of BMP, Indian Hedgehog, Notch and Wnt clustered according to the time postāfracture first and age second. Clinical Significance: Greater understanding of ageādependent molecular changes with healing will help form a mechanistic basis for therapies to improve patient outcomes. Ā© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:149ā158, 2018.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142531/1/jor23652.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142531/2/jor23652_am.pd
Suppression of Notch Signaling in Osteoclasts Improves Bone Regeneration and Healing
Owing to the central role of osteoclasts in bone physiology and remodeling, manipulation of their maturation process provides a potential therapeutic strategy for treating bone diseases. To investigate this, we genetically inhibited the Notch signaling pathway in the myeloid lineage, which includes osteoclast precursors, using a dominant negative form of MAML (dnMAML) that inhibits the transcriptional complex required for downstream Notch signaling. Osteoclasts derived from dnMAML mice showed no significant differences in early osteoclastic gene expression compared to the wild type. Further, these demonstrated significantly lowered resorption activity using bone surfaces while retaining their osteoblast stimulating ability using ex vivo techniques. Using in vivo approaches, we detected significantly higher bone formation rates and osteoblast gene expression in dnMAML cohorts. Further, these mice exhibited increased bone/tissue mineral density compared to wild type and larger bony calluses in later stages of fracture healing. These observations suggest that therapeutic suppression of osteoclast Notch signaling could reduce, but not eliminate, osteoclastic resorption without suppression of restorative bone remodeling and, therefore, presents a balanced paradigm for increasing bone formation, regeneration, and healing. Ā© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2089ā2103, 2019Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151846/1/jor24384.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151846/2/jor24384_am.pd
Canonical Notch signaling is required for bone morphogenetic proteināmediated human osteoblast differentiation
Osteoblast differentiation of bone marrowāderived 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 24āhours 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, siRNAāmediated ablation of RBPJ decreased BMPāmediated osteoblastogenesis. Finally, systemic Notch inhibition using diabenzazepine (DBZ) reduced BMP2āinduced calvarial bone healing in mice supporting the critical regulatory role of Notch signaling in BMPāinduced osteoblastogenesis.Bone morphogenetic protein (BMP) stimulation of boneāmarrowāderived human mesenchymal progenitor cells (hMSCs) increases Notch proteins via increased Notch ligand Jagged1 expression. Canonical Notch signaling is required for BMPāinduced ALPL expression and osteoblastic commitment of hMSCs. Both BMPāinduced osteoblastogenesis and Notchāinduced 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
Thrombospondin-2 Influences the Proportion of Cartilage and Bone During Fracture Healing
Thrombospondin-2 (TSP2) is a matricellular protein with increased expression during growth and regeneration. TSP2-null mice show accelerated dermal wound healing and enhanced bone formation. We hypothesized that bone regeneration would be enhanced in the absence of TSP2. Closed, semistabilized transverse fractures were created in the tibias of wildtype (WT) and TSP2-null mice. The fractures were examined 5, 10, and 20 days after fracture using Ī¼CT, histology, immunohistochemistry, quantitative RT-PCR, and torsional mechanical testing. Ten days after fracture, TSP2-null mice showed 30% more bone by Ī¼CT and 40% less cartilage by histology. Twenty days after fracture, TSP2-null mice showed reduced bone volume fraction and BMD. Mice were examined 5 days after fracture during the stage of neovascularization and mesenchymal cell influx to determine a cellular explanation for the phenotype. TSP2-null mice showed increased cell proliferation with no difference in apoptosis in the highly cellular fracture callus. Although mature bone and cartilage is minimal 5 days after fracture, TSP2-null mice had reduced expression of collagen IIa and Sox9 (chondrocyte differentiation markers) but increased expression of osteocalcin and osterix (osteoblast differentiation markers). Importantly, TSP2-null mice had a 2-fold increase in vessel density that corresponded with a reduction in vascular endothelial growth factor (VEGF) and Glut-1 (markers of hypoxia inducible factor [HIF]-regulated transcription). Finally, by expressing TSP2 using adenovirus starting 3 days after fracture, chondrogenesis was restored in TSP2-null mice. We hypothesize that TSP2 expressed by cells in the fracture mesenchyme regulates callus vascularization. The increase in vascularity increases tissue oxemia and decreases HIF; thus, undifferentiated cells in the callus develop into osteoblasts rather than chondrocytes. This leads to an alternative strategy for achieving fracture healing with reduced endochondral ossification and enhanced appositional bone formation. Controlling the ratio of cartilage to bone during fracture healing has important implications for expediting healing or promoting regeneration in nonunions
Editorial: Interdependencies and interfaces in bone regeneration ā the immune status at its core
Type III collagen modulates fracture callus bone formation and early remodeling
Type III collagen (Col3) has been proposed to play a key role in tissue repair based upon its temporospatial expression during the healing process of many tissues, including bone. Given our previous finding that Col3 regulates the quality of cutaneous repair, as well as our recent data supporting its role in regulating osteoblast differentiation and trabecular bone quantity, we hypothesized that mice with diminished Col3 expression would exhibit altered longābone fracture healing. To determine the role of Col3 in bone repair, young adult wildātype (Col3+/+) and haploinsufficent (Col3+/ā) mice underwent bilateral tibial fractures. Healing was assessed 7, 14, 21, and 28 days following fracture utilizing microcomputed tomography (microCT), immunohistochemistry, and histomorphometry. MicroCT analysis revealed a small but significant increase in bone volume fraction in Col3+/ā mice at day 21. However, histological analysis revealed that Col3+/ā mice have less bone within the callus at days 21 and 28, which is consistent with the established role for Col3 in osteogenesis. Finally, a reduction in fracture callus osteoclastic activity in Col3+/ā mice suggests Col3 also modulates callus remodeling. Although Col3 haploinsufficiency affected biological aspects of bone repair, it did not affect the regain of mechanical function in the young mice that were evaluated in this study. These findings provide evidence for a modulatory role for Col3 in fracture repair and support further investigations into its role in impaired bone healing. Ā© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:675ā684, 2015.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111249/1/jor22838.pd
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