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| 2. |
- Grundberg, Elin, et al.
(författare)
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Population genomics in a disease targeted primary cell model
- 2009
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Ingår i: Genome Research. - : Cold Spring Harbor Laboratory. - 1088-9051 .- 1549-5469. ; 19:11, s. 1942-1952
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Tidskriftsartikel (refereegranskat)abstract
- The common genetic variants associated with complex traits typically lie in noncoding DNA and may alter gene regulation in a cell type-specific manner. Consequently, the choice of tissue or cell model in the dissection of disease associations is important. We carried out an expression quantitative trait loci (eQTL) study of primary human osteoblasts (HOb) derived from 95 unrelated donors of Swedish origin, each represented by two independently derived primary lines to provide biological replication. We combined our data with publicly available information from a genome-wide association study (GWAS) of bone mineral density (BMD). The top 2000 BMD-associated SNPs (P < approximately 10(-3)) were tested for cis-association of gene expression in HObs and in lymphoblastoid cell lines (LCLs) using publicly available data and showed that HObs have a significantly greater enrichment (threefold) of converging cis-eQTLs as compared to LCLs. The top 10 BMD loci with SNPs showing strong cis-effects on gene expression in HObs (P = 6 x 10(-10) - 7 x 10(-16)) were selected for further validation using a staged design in two cohorts of Caucasian male subjects. All 10 variants were tested in the Swedish MrOS Cohort (n = 3014), providing evidence for two novel BMD loci (SRR and MSH3). These variants were then tested in the Rotterdam Study (n = 2090), yielding converging evidence for BMD association at the 17p13.3 SRR locus (P(combined) = 5.6 x 10(-5)). The cis-regulatory effect was further fine-mapped to the proximal promoter of the SRR gene (rs3744270, r(2) = 0.5, P = 2.6 x 10(-15)). Our results suggest that primary cells relevant to disease phenotypes complement traditional approaches for prioritization and validation of GWAS hits for follow-up studies.
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| 3. |
- Grundberg, Elin, et al.
(författare)
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Systematic assessment of the human osteoblast transcriptome in resting and induced primary cells
- 2008
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Ingår i: Physiological Genomics. - : American Physiological Society. - 1094-8341 .- 1531-2267. ; 33:3, s. 301-11
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Tidskriftsartikel (refereegranskat)abstract
- Osteoblasts are key players in bone remodeling. The accessibility of human primary osteoblast-like cells (HObs) from bone explants makes them a lucrative model for studying molecular physiology of bone turnover, for discovering novel anabolic therapeutics, and for mesenchymal cell biology in general. Relatively little is known about resting and dynamic expression profiles of HObs, and to date no studies have been conducted to systematically assess the osteoblast transcriptome. The aim of this study was to characterize HObs and investigate signaling cascades and gene networks with genomewide expression profiling in resting and bone morphogenic protein (BMP)-2- and dexamethasone-induced cells. In addition, we compared HOb gene expression with publicly available samples from the Gene Expression Omnibus. Our data show a vast number of genes and networks expressed predominantly in HObs compared with closely related cells such as fibroblasts or chondrocytes. For instance, genes in the insulin-like growth factor (IGF) signaling pathway were enriched in HObs (P = 0.003) and included the binding proteins (IGFBP-1, -2, -5) and IGF-II and its receptor. Another HOb-specific expression pattern included leptin and its receptor (P < 10(-8)). Furthermore, after stimulation of HObs with BMP-2 or dexamethasone, the expression of several interesting genes and pathways was observed. For instance, our data support the role of peripheral leptin signaling in bone cell function. In conclusion, we provide the landscape of tissue-specific and dynamic gene expression in HObs. This resource will allow utilization of osteoblasts as a model to study specific gene networks and gene families related to human bone physiology and diseases.
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| 5. |
- Kwan, Tony, et al.
(författare)
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Tissue effect on genetic control of transcript isoform variation.
- 2009
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Ingår i: PLoS genetics. - : Public Library of Science (PLoS). - 1553-7404 .- 1553-7390. ; 5:8
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Tidskriftsartikel (refereegranskat)abstract
- Current genome-wide association studies (GWAS) are moving towards the use of large cohorts of primary cell lines to study a disease of interest and to assign biological relevance to the genetic signals identified. Here, we use a panel of human osteoblasts (HObs) to carry out a transcriptomic survey, similar to recent studies in lymphoblastoid cell lines (LCLs). The distinct nature of HObs and LCLs is reflected by the preferential grouping of cell type-specific genes within biologically and functionally relevant pathways unique to each tissue type. We performed cis-association analysis with SNP genotypes to identify genetic variations of transcript isoforms, and our analysis indicates that differential expression of transcript isoforms in HObs is also partly controlled by cis-regulatory genetic variants. These isoforms are regulated by genetic variants in both a tissue-specific and tissue-independent fashion, and these associations have been confirmed by RT-PCR validation. Our study suggests that multiple transcript isoforms are often present in both tissues and that genetic control may affect the relative expression of one isoform to another, rather than having an all-or-none effect. Examination of the top SNPs from a GWAS of bone mineral density show overlap with probeset associations observed in this study. The top hit corresponding to the FAM118A gene was tested for association studies in two additional clinical studies, revealing a novel transcript isoform variant. Our approach to examining transcriptome variation in multiple tissue types is useful for detecting the proportion of genetic variation common to different cell types and for the identification of cell-specific isoform variants that may be functionally relevant, an important follow-up step for GWAS.
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| 6. |
- Marsell, Richard, et al.
(författare)
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GSK-3 inhibition by an orally active small molecule increases bone mass in rats
- 2012
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Ingår i: Bone. - : Elsevier BV. - 8756-3282 .- 1873-2763. ; 50:3, s. 619-627
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Tidskriftsartikel (refereegranskat)abstract
- Glycogen synthase kinase 3β (GSK-3β) actions are central in the canonical Wnt pathway, important in many biological processes and a potential drug target for treating several diseases. It is appreciated that a balanced Wnt canonical signaling is crucial for the maintenance of normal bone mass. In this study we investigated the effects of a potent orally active GSK-3 inhibitor, AZD2858, on bone mass in rats. Treatment (1μM) of human osteoblast cells with AZD2858 in vitro increased β-catenin levels after a short period of time. In rats, oral AZD2858 treatment caused a dose-dependent increase in trabecular bone mass compared to control after a two-week treatment with a maximum effect at a dose of 20mg/kg once daily (total BMC: 172% of control; p<0.001). A small but significant effect was also seen at cortical sites (total BMC: 111% of control; p<0.001). Biomechanical testing demonstrated an increase in both vertebral compression strength at a dose of 20mg/kg once daily (Load at failure: 370% of control, p<0.001) and diaphyseal strength of femora subjected to a three point bending test (Load at failure: 115% of control; p<0.01). Furthermore, histomorphometry showed a dramatic increase in bone formation indices, and serum markers of both bone formation (Osteocalcin, 146% of control; p<0.001) and resorption (CTX, 189% of control; p<0.001) were elevated. Our conclusion is that a GSK-3 inhibitor drug may prove effective as an anabolic strategy in the treatment of diseases characterized by low bone mass, since AZD2858 has extensive bone building effects at predominantly trabecular sites.
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| 7. |
- Silfverswärd, Carl-Johan, et al.
(författare)
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Bone formation in interleukin-4 and interleukin-13 depleted mice.
- 2008
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Ingår i: Acta orthopaedica. - : Medical Journals Sweden AB. - 1745-3682 .- 1745-3674. ; 79:3, s. 410-20
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND AND PURPOSE: Cytokines play an important role in the complex process of bone formation. We have previously found an altered skeletal phenotype with reduction of cortical bone mass in mice depleted of the 2 cytokines interleukin-4 (IL-4) and interleukin-13 (IL 13). The present study was performed to investigate a potential role of IL-4 and IL-13 in fracture healing and bone induction by demineralized xenogenic bone matrix (DXBM). METHODS: Callus formation in IL-4-(/)-IL-13-(/)- (IL-4/13 knockout) and wild-type (WT) male mice was compared using a standardized fracture model. The capacity of IL-4(-/-)IL-13(-/-) and WT male and female mice to form heterotopic bone was compared using intramuscular implants of DXBM. Bone formation and mechanical properties were evaluated by pQCT, ash weight, 3-point bending, radiology, and immunohistology. RESULTS: In the fracture investigation substantial amounts of new bone formation by 5 weeks were found, but no differences in radiographical healing, callus volume, BMD, BMC, or mechanical properties were detected between IL-4(-/-)IL-13(-/-) and WT mice. In the DXBM investigation radiographic analysis confirmed mineralization of implants in both groups, but no difference in the amount of mineral deposition (net bone formation) between IL-4(-/-)IL-13(-/-) and WT mice was found. Immunohistology showed inhibition of autonomic nerves in the capsule of the IL-4(-/-)IL-13(-/-) group along with a lack of vascularization within the implants. INTERPRETATION: Depletion of IL-4 and IL-13 does not cause any major alteration in fracture healing or heterotopic bone formation in mice. The pattern of autonomous nerve expression and expression of markers of neovascularization is, however, altered to some extent by the absence of IL-4 and IL-13.
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| 8. |
- Silfverswärd, Carl-Johan, 1961-
(författare)
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Effects of Interleukin-4 and Interleukin-13 on Bone
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cytokines play important roles in bone metabolism, participating in the complex interplay necessary for normal bone formation and turnover. The aim of the present thesis was to investigate the effects of two anti-inflammatory cytokines, interleukin-4 (IL-4) and interleukin-13 (IL-13) on bone. Influence of pro- and anti-inflammatory cytokines on interleukin-6 (IL-6) formation in cultured human osteoblasts (hOBs) was investigated. IL-4 and IL-13 as well as interleukin-1 (IL-1) and tumour necrosis factor alpha and beta (TNF-α/β) stimulated IL-6 secretion in hOBs. Also, IL-4 and IL-13 synergistically potentiated the effect of IL-1 and TNFs on IL-6 secretion. Effects of IL-4 and IL-13 on markers of osteoblastic activity in hOBs were investigated. IL-4 and IL-13 induced a dose-dependent increase in the formation of alkaline phosphatase (ALP) and pro-collagen type I carboxy-peptide (PICP) together with enhanced mineralization rate in hOBs. Formation of osteocalcin (OC) was unaffected. The mechanism behind inhibited proliferation by IL-4 and IL-13 in hOBs was investigated. IL-4 and IL-13 caused a dose-dependent increase in DNA-fragmentation together with escalating Caspase-3 activity in hOBs, reflecting induced apoptosis. Osteoblast apoptosis was also confirmed by TNF-α, dexamethasone and by serum starvation.The skeletal phenotype of IL-13-/-, IL-4-/-IL-13-/- and WT mice was compared. An altered cortical bone mass was detected in adult male IL-4-/-IL-13-/- mice. They displayed a reduction in cortical bone mineral content (BMC) secondary to reduced cortical thickness. Mechanical strength of the cortical bone was reduced in level with the reduction detected in BMC. Trabecular bone mineral density (tvBMD) was unaffected. Callus formation in IL-4-/-IL-13-/- and WT male mice was compared. No differences were found concerning radiological healing, biomechanical properties, callus parameters or histology. Heterotopic bone formation in IL-4-/-IL-13-/- and WT mice was compared using DXBM implants. No differences were found concerning mineralization of implants. Immuno-histology showed inhibition of autonomic nerves and lack of implant vascularization in IL-4-/-IL-13-/- mice. In summery, the two anti-inflammatory cytokines IL-4 and IL-13 influence osteoblast activity and apoptosis in vitro. They also selectively influence cortical bone formation in vivo. These findings suggest a role for IL-4 and IL-13 in osteoblast differentiation, in bone metabolism and in bone formation.
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| 9. |
- Silfverswärd, Carl-Johan, et al.
(författare)
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Expression of Markers of Activity in Cultured Human Osteoblasts : Effects of Interleukin-4 and Interleukin-13
- 2010
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Ingår i: Scandinavian Journal of Clinical and Laboratory Investigation. - : Informa UK Limited. - 0036-5513 .- 1502-7686. ; 70:5, s. 338-342
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Tidskriftsartikel (refereegranskat)abstract
- Cytokines regulate proliferation, differentiation and activation of osteoblasts. Interleukin-4 (IL-4) and interleukin-13 (IL-13) takes part in this regulation by inhibiting proliferation and by enhancement of interleukin-6 (IL-6) formation in cultured human osteoblasts (hOBs). In the present study we have investigated the effects of IL-4 and IL-13 on markers of osteoblastic activity in isolated hOBs. Treatment with either IL-4 or IL-13 (1–100 pM) stimulated the formation of alkaline phosphatase (ALP) dose-dependently, detected by enzyme reaction and histochemistry. IL-4 and IL-13 also induced an increase in the secretion of procollagen type I carboxypeptide (PICP) from cultured hOBs, measured by RIA. Osteocalcin secretion measured by ELISA-technique was unaffected. The rate of mineralization, assessed by von Kossa and Alizarin Red staining, was clearly enhanced in hOBs stimulated by IL-4 or IL-13. In conclusion IL-4 and IL-13 exert multiple effects on osteoblast activity in cultured hOBs. Stimulation of ALP secretion together with enhanced collagen secretion and mineralization suggests that IL-4 and IL-13 also have the capacity to maintain hOBs in a differentiated, productive phase.
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