The mechanisms underlying the inverse relationship between osteogenic and adipogenic differentiation of bone marrow stromal cells (MSC) are not known in detail. We have previously established two ...cell lines from mouse bone marrow that are committed to either osteogenic (osteoblasts and chondrocytes) (mMSC
Bone) or adipogenic (mMSC
Adipo) lineage. To identify the molecular mechanism determining the lineage commitment, we compared the basal gene expression profile of mMSC
Bone versus mMSC
Adipo using Affymetrix GeneChip® MG430A 2.0 Array. Gene annotation analysis based on biological function revealed an over-representation of skeletal development genes in mMSC
Bone while genes related to lipid metabolism and immune response were highly expressed in mMSC
Adipo. In addition, there was a significant up-regulation of canonical Wnt signalling genes in mMSC
Bone compared to mMSC
Adipo (
p
<
0.006). Dual-luciferase assay and expression analysis of genes related to Wnt signalling demonstrated significant activation of Wnt signalling pathway in mMSC
Bone compared to mMSC
Adipo. Reduced Wnt activity in mMSC
Adipo was associated with increased expression of the Wnt inhibitor, secreted frizzled-related protein 1 (sFRP-1) at both mRNA and protein levels in mMSC
Adipo. Interestingly, conditioned medium (CM) collected from mMSC
Adipo (mMSC-CM
Adipo) inhibited osteoblast differentiation of mMSC, while depletion of sFRP-1 protein from mMSC-CM
Adipo abolished its inhibitory effect on osteoblast differentiation. Furthermore, treatment of mMSC with recombinant sFRP-1 resulted in a dose-dependent inhibition of osteoblast and stimulation of adipocyte differentiation. In conclusion, cross-talk exists between different populations of MSC in the bone marrow, and Wnt signalling functions as a molecular switch that determines the balance between osteoblastogenesis and adipogenesis.
Fractures still present a significant burden to patients due to pain and periods of unproductivity. Numerous growth factors have been identified to regulate bone remodeling. However, to date, only ...the bone morphogenetic proteins (BMPs) are used to enhance fracture healing in clinical settings. Activins are pleiotropic growth factors belonging to the TGF-β superfamily. We and others have recently shown that treatment with recombinant fusion proteins of activin receptors greatly increases bone mass in different animal models by trapping activins and other ligands thus inhibiting their signaling pathways. However, their effects on fracture healing are less known. Twelve-week old male C57Bl mice were subjected to a standardized, closed tibial fracture model. Animals were divided into control and treatment groups and were administered either PBS control or a soluble activin type IIB receptor (ActRIIB-Fc) intraperitoneally once a week for a duration of two or four weeks. There were no significant differences between the groups at two weeks but we observed a significant increase in callus mineralization in ActRIIB-Fc-treated animals by microcomputed tomography imaging at four weeks. Bone volume per tissue volume was 60%, trabecular number 55% and bone mineral density 60% higher in the 4-week calluses of the ActRIIB-Fc-treated mice (p<0.05 in all). Biomechanical strength of 4-week calluses was also significantly improved by ActRIIB-Fc treatment as stiffness increased by 64% and maximum force by 45% (p<0.05) compared to the PBS-injected controls. These results demonstrate that ActRIIB-Fc treatment significantly improves healing of closed long bone fractures. Our findings support the previous reports of activin receptors increasing bone mass but also demonstrate a novel approach for using ActRIIB-Fc to enhance fracture healing.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Exosomes participate in intercellular messaging by transporting bioactive lipid-, protein- and RNA-molecules and -complexes. The contents of the exosomes reflect the physiological status of an ...individual making exosomes promising targets for biomarker analyses. In the present study we extracted exosome microRNAs (exomiRs) from serum samples of premenopausal women (n = 8) and monozygotic postmenopausal twins (n = 10 female pairs), discordant for the use of estrogenic hormone replacement therapy (HRT), in order to see whether the age or/and the use of HRT associates with exomiR content. A total of 241 exomiRs were detected by next generation sequencing, 10 showing age, 14 HRT and 10 age +HRT -related differences. When comparing the groups, differentially expressed miRs were predicted to affect cell proliferation processes showing inactivation with younger age and HRT usage. MiR-106-5p, -148a-3p, -27-3p, -126-5p, -28-3p and -30a-5p were significantly associated with serum 17β-estradiol. MiRs formed two hierarchical clusters being indicative of positive or negative health outcomes involving associations with body composition, serum 17β-estradiol, fat-, glucose- and inflammatory markers. Circulating exomiR clusters, obtained by NGS, could be used as indicators of metabolic and inflammatory status affected by hormonal changes at menopause. Furthermore, the individual effects of HRT-usage could be evaluated based on the serum exomiR signature.
Fracture healing is a complex process with multiple overlapping metabolic and differentiation phases. Small non-coding RNAs are involved in the regulation of fracture healing and their presence in ...circulation is under current interest due to their obvious value as potential biomarkers. Circulating microRNAs (miRNAs) have been characterized to some extent but the current knowledge on tRNA-derived small RNA fragments (tsRNAs) is relatively scarce, especially in circulation.
In this study, the spectrum of circulating miRNAs and tsRNAs was analysed by next generation sequencing to show their differential expression during fracture healing in vivo. Analysed tsRNA fragments included stress-induced translation interfering tRNA fragments (tiRNAs or tRNA halves) and internal tRNA fragments (i-tRF), within the size range of 28–36 bp. To unveil the expression of these non-coding RNAs, genome-wide analysis was performed on two months old C57BL/6 mice on days 1, 5, 7, 10, and 14 (D1, D5, D7, D10, and D14) after a closed tibial fracture.
Valine isoacceptor tRNA-derived Val-AAC 5′end and Val-CAC 5′end fragments were the major types of 5′end tiRNAs in circulation, comprising about 65 % of the total counts. Their expression was not affected by fracture. After a fracture, the levels of two 5′end tiRNAs Lys-TTT 5′ and Lys-CTT 5′ were decreased and His-GTG 5′ was increased through D1-D14. The level of miR-451a was decreased on the first post-fracture day (D1), whereas miR-328-3p, miR-133a-3p, miR-375-3p, miR-423-5p, and miR-150-5p were increased post-fracture. These data provide evidence on how fracture healing could provoke systemic metabolic effects and further pinpoint the potential of small non-coding RNAs as biomarkers for tissue regeneration.
Chondrocyte differentiation is a principal progress in endochondral ossification and in the formation of secondary ossification center (SOC) during the long bone development. We have previously ...reported that targeted deletion of Wnt1 in mesenchymal progenitors (Wnt1Prrx−/−) leads to spontaneous fractures and severe osteopenia in mouse long bones, suggesting that Wnt1 is a key regulator of bone metabolism. However, the effect of Wnt1 on the regulation of cartilage development and chondrocyte differentiation remained unknown. In this study, WNT1 protein expression was observed in lateral superficial cartilage and growth plate pre-hypertrophic chondrocytes in mice. Wnt1 mRNA expression was detected in epiphyseal cartilage from E16.5 to 3 month-old mice. Detailed histological analyses revealed that the average thickness and chondrocyte density of proximal tibial articular cartilage and growth plate were unchanged between Wnt1Prrx−/− and control mice. However, μCT analysis of tibial epiphyses showed that the subchondral bone mass was reduced in Wnt1Prrx−/− mice compared to control mice, as demonstrated by decreased bone volume, trabecular number, trabecular thickness, and increased trabecular separation in Wnt1Prrx−/− mice. Mechanistically, histomorphometric analyses showed that the reduced subchondral bone mass in Wnt1Prrx−/− mice was due to impaired bone formation and enhanced bone resorption. In vitro, exogenous Wnt1 inhibited chondrogenesis and chondrocyte hypertrophy in both cell autonomous and juxtacrine manners, while matrix mineralization and the expression of Mmp13, Mmp9 and Opn were induced in a juxtacrine manner. Taken together, mesenchymal cell-derived Wnt1 is an important regulator of subchondral bone remodeling, although it has no effect on the regulation of growth plate or articular cartilage.
•Wnt1 has no effect on growth plate or articular cartilage.•Wnt1 is a key regulator of subchondral bone remodeling.•Wnt1 inhibits chondrogenesis and chondrocyte hypertrophy in vitro.
To circumvent the problems of genetic and environmental diversity hampering the analysis in humans, we turned to a murine model for human knee osteoarthritis (OA) and fine mapped a previously defined ...OA-quantitative trait locus (QTL). We here focused on one of the candidate genes within the OA-QTL encoding the Wnt antagonist secreted frizzled related protein 1 (Sfrp1). Sequence analysis of the Sfrp1 gene in the OA strain STR/ort revealed 23 polymorphic changes with a potential to alter the gene expression. Indeed, a reduced expression in STR/ort mice was demonstrated for articular chondrocytes and hypertrophic chondrocytes of the femoral growth plate as shown by immunohistochemistry. RT-PCR of in vitro generated mesenchymal stem cells (MSC) and chondrogenically differentiated MSC (cMSC) confirmed the reduced Sfrp1 expression in STR/ort mice. This reduced Sfrp1 expression in MSC correlated with an increased amount of cytoplasmic β-catenin, a downregulation of the Wnt target gene PPARγ and an upregulation of Runx2 as well as a preferential differentiation of the MSC along the osteoblasts lineage. Given the role of Wnt signalling during chondrogenesis and in maintaining the integrity of the long lived articular chondrocytes, we conclude from our results that the reduced Sfrp1 expression in STR/ort mice not only leads to an increased activation of the Wnt/β-catenin signalling early in life but also renders the articular cartilage prone to premature ageing and to the development of OA.
► A quantitative trait locus for osteoarthritis on the mouse chromosome 8 encodes Sfrp1. ► Sequence of the Sfrp1 gene in the STR/ort mouse suggests regulatory polymorphisms. ► Sfrp1 expression is decreased in STR/ort chondrocytes and mesenchymal stem cells (MSC). ► Wnt signalling is upregulated in STR/ort MSC. ► Altered Wnt signalling correlates with premature ageing of the chondrocytes.
Delta like‐1 (Dlk1)/preadipocyte factor‐1 (Pref‐1)/fetal antigen‐1 (FA1) is a novel surface marker for embryonic chondroprogenitor cells undergoing lineage progression from proliferation to ...prehypertrophic stages. However, mechanisms mediating control of its expression during chondrogenesis are not known. Thus, we examined the effect of a number of signaling molecules and their inhibitors on Dlk1 expression during in vitro chondrogenic differentiation in mouse embryonic limb bud mesenchymal micromass cultures and mouse embryonic fibroblast (MEF) pellet cultures. Dlk1/Pref‐1 was initially expressed during mesenchymal condensation and chondrocyte proliferation, in parallel with expression of Sox9 and Col2a1, and was downregulated upon the expression of Col10a1 by hypertrophic chondrocytes. Among a number of molecules that affected chondrogenesis, transforming growth factor‐β1 (TGF‐β1)‐induced proliferation of chondroprogenitors was associated with decreased Dlk1 expression. This effect was abolished by TGF‐β signaling inhibitor SB431542, suggesting regulation of Dlk1/FA1 by TGF‐β1 signaling in chondrogenesis. TGF‐β1‐induced Smad phosphorylation and chondrogenesis were significantly increased in Dlk1−/− MEF, while they were blocked in Dlk1 overexpressing MEF, in comparison with wild‐type MEF. Furthermore, overexpression of Dlk1 or addition of its secreted form FA1 dramatically inhibited TGF‐β1‐induced Smad reporter activity. In conclusion, our data identified Dlk1/FA1 as a downstream target of TGF‐β1 signaling molecule that mediates its function in embryonic chondrogenesis. The crosstalk between TGF‐β1 and Dlk1/FA1 was shown to promote early chondrogenesis during the embryonic endochondral ossification process. STEM CELLS 2012; 30:304–313.
The RECQL4 helicase gene is a member of the RECQL gene family, mutated in some Rothmund–Thomson syndrome (RTS) patients. Other members of this gene family are BLM mutated in Bloom syndrome, WRN ...mutated in Werner syndrome and RECQL and RECQL5. All polypeptides encoded by RECQL genes share a central region of seven helicase domains. The function of RECQL4 remains unknown, but based on the domain homology it possesses ATP-dependent DNA helicase activity such as BLM and WRN. Rothmund–Thomson, Bloom and Werner syndromes have overlapping clinical features, of which high predisposition to malignancies is the most remarkable feature. Here we report a fourth syndrome resulting in mutations in the RECQL genes. RAPADILINO syndrome is an autosomal recessive disorder characterized by short stature, radial ray defects and other malformations, as well as infantile diarrhoea, but not by a significant cancer risk. Four mutations in the RECQL4 gene were found in the Finnish patients, the most common mutation representing exon 7 in-frame deletion saving the helicase domain and showing dominant effect over other three nonsense mutations. The tissue expression of Recql4 in mouse well agrees with the tissue symptoms of RAPADILINO. The skeletal malformations in RAPADILINO and RTS patients as well as the high osteosarcoma risk in RTS propose a special role for RECQL4 in bone development.
Delta-like 1 (Dlk1, also known as fetal antigen-1, FA1) is a member of Notch/Delta family that inhibits adipocyte and osteoblast differentiation; however, its role in chondrogenesis is still not ...clear. Thus, we overexpressed Dlk1/FA1 in mouse embryonic ATDC5 cells and tested its effects on chondrogenic differentiation. Dlk1/FA1 inhibited insulin-induced chondrogenic differentiation as evidenced by reduction of cartilage nodule formation and gene expression of aggrecan, collagen Type II and X. Similar effects were obtained either by using Dlk1/FA1-conditioned medium or by addition of a purified, secreted, form of Dlk1 (FA1) directly to the induction medium. The inhibitory effects of Dlk1/FA1 were dose-dependent and occurred irrespective of the chondrogenic differentiation stage: proliferation, differentiation, maturation, or hypertrophic conversion. Overexpression or addition of the Dlk1/FA1 protein to the medium strongly inhibited the activation of Akt, but not the ERK1/2, or p38 MAPK pathways, and the inhibition of Akt by Dlk1/FA1 was mediated through PI3K activation. Interestingly, inhibition of fibronectin expression by siRNA rescued the Dlk1/FA1-mediated inhibition of Akt, suggesting interaction of Dlk1/FA1 and fibronectin in chondrogenic cells. Our results identify Dlk1/FA1 as a novel regulator of chondrogenesis and suggest Dlk1/FA1 acts as an inhibitor of the PI3K/Akt pathways that leads to its inhibitory effects on chondrogenesis.
Chondrogenic differentiation in mesenchymal stromal cells (MSCs) has been actively studied due to their potential use in mesenchymal tissue repair. Our goal was to develop a simple isolation protocol ...for adherent mouse MSCs to simultaneously clear off hematopoietic cells and expand to obtain enough starting material for differentiation studies. CD34 and CD45 expressing cells were rapidly removed by inhibiting growth of hematopoietic cells to yield short-term selected (STS) cells. Further passaging enriched more primitive, uniformly Sca-1 expressing, long-term selected (LTS) cells. The efficacy of several BMPs to induce chondrogenesis in pellet culture was compared in STS and LTS cells. In STS cells, chondrogenesis progressed rapidly to terminal differentiation while LTS cells differentiated at a slower rate with no hypertrophy. In LTS cells, rhBMP homodimers -2, -4, -6 and rhBMP2/7 heterodimer were effective enhancers of chondrogenesis over that of rhBMP-5 and -7. In STS cells, rhBMP-2 and rhBMP-7 supported rapid chondrogenesis and terminal differentiation over that of rhBMP-6. These data indicate the impact of stromal cell composition on the chondrogenic differentiation profile, which is an important aspect to be considered when standardizing differentiation assay conditions as well as developing MSC based cartilage repair technologies.
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