Activin structure and activin signaling pathway. (A) Activins are a formed by dimerization of two inhibin ß subunits linked by a disulfide bond. (B) Activins from the circulation or produced locally in bone by the osteoblasts binds to a TGF-β type II serine/threonine kinase activity receptor (ACVR2A or ACVR2B). This binding leads to the recruitment and subsequent phosphorylation of a type I receptor (ACVR1B). The receptor complex is now active and phosphorylates Smad2 or 3 proteins (Smad2/3). Upon phosphorylation, Smad2/3 binds to Smad 4 and the Smad protein complex translocate into the nucleus where it binds transcription factors (TF) to regulate the expression of target genes.

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... are members of the TGF-β superfamily. In structural terms, activins are the result of homo-dimerization of two inhibin ß subunits linked by a disulfide bond [101] ( Figure 4A. These protein dimers exert their action via TGF-β signaling, that starts with the binding of the ligands to type I and II serine/threonine kinase receptors located on the cell surface. ...

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... protein dimers exert their action via TGF-β signaling, that starts with the binding of the ligands to type I and II serine/threonine kinase receptors located on the cell surface. Activins signal using the type II receptors ACVR2A or ACVR2B (also known as ActRII or ActRIIB) and the type I receptor ACVR1B ( Figure 4B). Upon ligand binding the type II receptor phosphorylates serine residues of the type I receptor, activating the receptor complex. ...

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... signal is transmitted further through Smad proteins, Smad2/3 and Smad4 in the case of activin [102][103] . Once phosphorylated, the active Smad proteins translocate to the nucleus binding to transcription factors and modulating the expression of target genes [104][105] (Figure 4B). ...

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... verified that most of the (FT-ICR-MS) quantified peptides, mapping to the same protein, have similar expression patterns ( Figure 4A). In order to validate the MS data, we selected two differentially expressed proteins that have been reported to be relevant for bone biology, ANXA2 and FN1. ...

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... order to validate the MS data, we selected two differentially expressed proteins that have been reported to be relevant for bone biology, ANXA2 and FN1. The expression pattern of these proteins was similar for both FT-ICR-MS and western blotting ( Figure 4A and 4B). We have also performed immunocytochemistry for FN1. ...

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... have also performed immunocytochemistry for FN1. As expected, FN1 immunocytochemistry from mineralized day 19 osteoblasts disclosed a clear extracellular localization of this protein ( Figure 4C). ...

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... (FN1, LGALS1) and calcium binding (ANXA2, ANXA1) proteins were also identified among differentially expressed proteins. FN1 is an abundant ECM glycoprotein with significantly higher expression, at all timepoints analyzed, in differentiating osteoblasts relative to their non-differentiated counterparts ( Figure 4A and 4B). FN1 is required for osteoblast differentiation and mineralization through interaction with the integrin a5β1 FN1 receptor [37] . ...

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... of total MVs and ALP positive MVs (ALP+ MVs) was determined using FACS. The production of MVs, total and ALP+, did not differ between vehicle and activin A conditions before the onset of mineralization ( Figure 4A). However, at the onset of mineralization the production of ALP+ MVs in vehicle osteoblasts increased sharply whereas in activin A condition this was significantly suppressed (Figure 4A). ...

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... production of MVs, total and ALP+, did not differ between vehicle and activin A conditions before the onset of mineralization ( Figure 4A). However, at the onset of mineralization the production of ALP+ MVs in vehicle osteoblasts increased sharply whereas in activin A condition this was significantly suppressed (Figure 4A). At the onset of mineralization, only 8% of the total MVs were ALP+ in the activin A condition compared to the 72% in the vehicle condition ( Figure 4A and 4B). ...

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... at the onset of mineralization the production of ALP+ MVs in vehicle osteoblasts increased sharply whereas in activin A condition this was significantly suppressed (Figure 4A). At the onset of mineralization, only 8% of the total MVs were ALP+ in the activin A condition compared to the 72% in the vehicle condition ( Figure 4A and 4B). In summary, our results indicate a bi- modal effect of activin A by changing the ECM composition and suppressing the provision of the biomineralization initiators, the MVs. ...

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... have also developed an emPAI-sum score as a measure for the impact of these functionally related protein groups, highlighting important biological mechanisms in the samples analyzed. The results for the emPAI-sum score application to our bone samples is illustrated in Figure 4. As an example, 98 transporter proteins were detected in bone and the sum of their emPAI scores was 247. ...

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... the protein families, following transporters at distance, appear 65 proteins with peptidase activity and an emPAI-sum of 38.1. Besides transcription regulators (28 proteins), kinases (26), transmembrane receptors (23), ion channels (11) we have also detected 3 cytokines (chromosome 19 open reading frame 10; complement component 5; secreted phosphoprotein 1) and 4 growth factors (C-type lectin domain family 11, member A; glia maturation factor, beta; hepatoma-derived growth factor; osteoglycin), with an emPAI-sum of 1.30 and 0.70 respectively ( Figure 4A). ...

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... among the most abundant protein families was the one annotated with GO term GO:0000786 (i.e. nucleosome) and an emPAI-sum of 212, well above the 71.0 obtained for ECM (GO:0031012) proteins ( Figure 4B). We further excluded the hypothesis that the high abundance given by the emPAI scores for nucleosome proteins (including histones) would be a consequence of bias in the protein extraction method, favoring alkaline proteins such as histones (Supplementary Figure II, Chapter 7). ...

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... over-represented terms having high emPAI-sums were pyrophosphatase activity (GO:0016462; emPAI-sum of 180), Ca 2+ -dependent phospholipid binding (GO:0005544; 49.7), antioxidant activity (GO:0016209; 46.1), integrin (GO:0008305; 3.73) and laminin (GO:0043256; 1.48) complex proteins. The highest abundant proteins within the nucleosome, Ca 2+ -dependent phospholipid binding and antioxidant activity GO-terms are shown in Figure 4C. Besides being significantly over-represented, these three groups based on these three GO terms also contain many of the most abundant proteins. ...

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... the direction of gene expression regulation, 1968 probes were identically regulated while 150 were oppositely changed during CVC and osteoblast development (Supplementary Table IV). The temporal and directional expression dynamics of the 4782 differential expressed probes during CVC development and osteoblast differentiation is resumed in Figure 4. K-means clustering separated the differentially expressed probes during CVC development and osteoblast differentiation into clusters sharing common regulation patterns. ...

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... temporal and directional expression dynamics of the 4782 differential expressed probes during CVC development and osteoblast differentiation is resumed in Figure 4. K-means clustering separated the differentially expressed probes during CVC development and osteoblast differentiation into clusters sharing common regulation patterns. On basis of Figure of Merit (FOM) analysis we concluded to divide gene expression data in 6 clusters ( Figure 4A). This number of clusters was found to provide good predictive power for the k-means algorithm (Supplementary Figure III, Chapter 7) without restricting the cluster size for functional annotation analysis. ...

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... number of clusters was found to provide good predictive power for the k-means algorithm (Supplementary Figure III, Chapter 7) without restricting the cluster size for functional annotation analysis. Functional GO annotation of genes underlying these clusters revealed information about the biological processes, cellular compartments and molecular functions during CVC development and osteoblast differentiation ( Figure 4B). ...

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... 1, 2 and 3 contained up-regulated genes while clusters 4, 5 and 6 represented down-regulated genes in both CVCs and osteoblasts ( Figure 4A). In clusters 1 and 2 CVCs and osteoblast shared the over-representation of genes linked to extracellular region (GO:0044421 and GO:0005576, Figure 4B). ...

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... 1, 2 and 3 contained up-regulated genes while clusters 4, 5 and 6 represented down-regulated genes in both CVCs and osteoblasts ( Figure 4A). In clusters 1 and 2 CVCs and osteoblast shared the over-representation of genes linked to extracellular region (GO:0044421 and GO:0005576, Figure 4B). In clusters 3, 4, and 5 several GO-terms were also shared by CVCs and osteoblasts but these were more general GO-terms like cell cycle, RNA processing, chromosome, biological response to organic substance, etc., related to general cell function/metabolism. ...

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... from cluster 1 and 2 ( Figure 4A and 4B) was similarly regulated by both cell types indicating a shared mechanism involving changes in the extracellular environment/matrix. ...

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... these, we identified calcium-binding proteins to be among the most over-represented in the bone formation proteome (Table 1). Annexins are a particularly relevant example, being abundant in human bone tissue (Chapter 4, Figure 4) and regulated during osteoblast differentiation (Chapter 2, Table 2). These proteins are key factors for MV-mediated mineralization, promoting the influx of calcium into these vesicles [9][10][11] during the last stages of osteoblast differentiation. ...

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... and phosphatases represent another class of proteins important for mineralization providing the phosphate that conjugates with calcium for hydroxyapatite formation. Despite not represented in Table 1, many pyrophosphatases were high abundant in the bone tissue (Chapter 4, Figure 4). ...

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... to our proteome data, osteoblasts and bone appear to be well prepared to control the levels of ROS. During osteoblast differentiation antioxidant proteins were regulated (Chapter 2, Table 2), proteins that were also found in bone tissue as high abundant (Chapter 4, Figure 4). Despite highlighting antioxidant proteins as proteins that fit particularly well in a role to preserve bone tissue integrity, we do not exclude their direct involvement in the regulation of bone formation [20][21] or resorption [22][23] . ...

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... bone tissue proteome (Chapter 4) shows that this definition can be misleading and depreciative for the NCP content of bone. Even though it represents only 10% of total bone protein content, the range of NCPs identified is vast (1213 proteins; Chapter 4, Figure 4). Furthermore, osteoblasts were not the unique source of NCPs in trabecular bone. ...

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... A is responsible for the inhibition of osteoblast differentiation and mineralization [31][32] . In Chapter 3 we extended the comprehension about the activin A effects in osteoblast differentiation demonstrating that activin signaling targets ECM maturity (Figure 2 and Table 1) disturbing the subsequent mineralization period with impairment of MV secretion (Figure 4). ...