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Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation.

Chiellini C, Cochet O, Negroni L, Samson M, Poggi M, Ailhaud G, Alessi MC, Dani C, Amri EZ - BMC Mol. Biol. (2008)

Bottom Line: Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly.Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2.Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome.

View Article: PubMed Central - HTML - PubMed

Affiliation: ISBDC, Université de Nice Sophia-Antipolis, CNRS ; 28 avenue de Valrose, 06100 Nice, France. Chiara.Chiellini@gmail.com

ABSTRACT

Background: It is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS) at an early step of commitment to adipocytes and osteoblasts.

Results: A proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 73 proteins at day 0 and day 3 of adipocyte and osteoblast differentiation. Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly. These proteins were classified into 8 clusters according to their function. Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2. Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome. Furthermore we showed that PAI-1 mRNA was down-regulated in the bone of ovariectomized mice.

Conclusion: Given its regulation during the early events of hMADS cell differentiation and its status in ovariectomized mice, PAI-1 could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis.

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Evaluation of the presence of the plasminogen system in hMADS cells. (A) PAI-1 mRNA levels have been determined in hMADS cells at day 0, day 3 adipocytes (adipo) and day 3 osteoblasts (osteo). (B) PAI-1 and (C) tPA protein levels have been measured by ELISA in the secretion media of hMADS cells at day 0, day 3 adipocytes (adipo) and day 3 osteoblasts (osteo) after 6 h of incubation. *: p < 0.05. (D) Zymographic analysis of plasminogen activators activity in hMADS cell conditioned media collected after 6 h of incubation. A representative casein-plasminogen zymogram out of three independent experiments is shown. (E) RT-PCR analysis of UPAR expression in hMADS cells at day 0, day 3 adipocytes and day 3 osteoblasts. β-actin expression is reported as internal control. PCR products have been separated on a 1% agarose gel.
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Figure 5: Evaluation of the presence of the plasminogen system in hMADS cells. (A) PAI-1 mRNA levels have been determined in hMADS cells at day 0, day 3 adipocytes (adipo) and day 3 osteoblasts (osteo). (B) PAI-1 and (C) tPA protein levels have been measured by ELISA in the secretion media of hMADS cells at day 0, day 3 adipocytes (adipo) and day 3 osteoblasts (osteo) after 6 h of incubation. *: p < 0.05. (D) Zymographic analysis of plasminogen activators activity in hMADS cell conditioned media collected after 6 h of incubation. A representative casein-plasminogen zymogram out of three independent experiments is shown. (E) RT-PCR analysis of UPAR expression in hMADS cells at day 0, day 3 adipocytes and day 3 osteoblasts. β-actin expression is reported as internal control. PCR products have been separated on a 1% agarose gel.

Mentions: Given the pattern of expression at the early step of adipogenesis and osteogenesis of hMADS cells, PAI-1 appeared as one of the most promising candidates for investigating further the adipocyte/osteoblast balance. PAI-1 mRNA levels were similar to those of protein levels (Fig. 5A). Since PAI-1 is a key regulator of the conversion of plasminogen to plasmin, negatively acting on two plasminogen activators, namely uPA (urinary plasminogen activator) and tPA (tissue type plasminogen activator) [24], we decided to evaluate uPA and tPA levels in hMADS cell secretome. ELISA experiments were performed on secretion media from hMADS cells at day 0 and day 3 under adipogenic and osteogenic differentiating conditions after 6 h of incubation. As reported in Figure 5B, consistent with the immunoblotting data, PAI-1 levels strongly decreased in differentiating adipocytes as compared to day 0. By contrast, PAI-1 was secreted by day 3 osteoblasts, at albeit the same levels when compared to day 0. Concerning tPA measurement, we did not detect tPA in day 3 differentiating adipocytes, while a significant increase of tPA levels was observed in day 3 osteoblasts as compared to day 0 (Fig. 5C). By contrast, uPA remained undetectable in the secretion media of hMADS cells under the three conditions analyzed.


Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation.

Chiellini C, Cochet O, Negroni L, Samson M, Poggi M, Ailhaud G, Alessi MC, Dani C, Amri EZ - BMC Mol. Biol. (2008)

Evaluation of the presence of the plasminogen system in hMADS cells. (A) PAI-1 mRNA levels have been determined in hMADS cells at day 0, day 3 adipocytes (adipo) and day 3 osteoblasts (osteo). (B) PAI-1 and (C) tPA protein levels have been measured by ELISA in the secretion media of hMADS cells at day 0, day 3 adipocytes (adipo) and day 3 osteoblasts (osteo) after 6 h of incubation. *: p < 0.05. (D) Zymographic analysis of plasminogen activators activity in hMADS cell conditioned media collected after 6 h of incubation. A representative casein-plasminogen zymogram out of three independent experiments is shown. (E) RT-PCR analysis of UPAR expression in hMADS cells at day 0, day 3 adipocytes and day 3 osteoblasts. β-actin expression is reported as internal control. PCR products have been separated on a 1% agarose gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2279142&req=5

Figure 5: Evaluation of the presence of the plasminogen system in hMADS cells. (A) PAI-1 mRNA levels have been determined in hMADS cells at day 0, day 3 adipocytes (adipo) and day 3 osteoblasts (osteo). (B) PAI-1 and (C) tPA protein levels have been measured by ELISA in the secretion media of hMADS cells at day 0, day 3 adipocytes (adipo) and day 3 osteoblasts (osteo) after 6 h of incubation. *: p < 0.05. (D) Zymographic analysis of plasminogen activators activity in hMADS cell conditioned media collected after 6 h of incubation. A representative casein-plasminogen zymogram out of three independent experiments is shown. (E) RT-PCR analysis of UPAR expression in hMADS cells at day 0, day 3 adipocytes and day 3 osteoblasts. β-actin expression is reported as internal control. PCR products have been separated on a 1% agarose gel.
Mentions: Given the pattern of expression at the early step of adipogenesis and osteogenesis of hMADS cells, PAI-1 appeared as one of the most promising candidates for investigating further the adipocyte/osteoblast balance. PAI-1 mRNA levels were similar to those of protein levels (Fig. 5A). Since PAI-1 is a key regulator of the conversion of plasminogen to plasmin, negatively acting on two plasminogen activators, namely uPA (urinary plasminogen activator) and tPA (tissue type plasminogen activator) [24], we decided to evaluate uPA and tPA levels in hMADS cell secretome. ELISA experiments were performed on secretion media from hMADS cells at day 0 and day 3 under adipogenic and osteogenic differentiating conditions after 6 h of incubation. As reported in Figure 5B, consistent with the immunoblotting data, PAI-1 levels strongly decreased in differentiating adipocytes as compared to day 0. By contrast, PAI-1 was secreted by day 3 osteoblasts, at albeit the same levels when compared to day 0. Concerning tPA measurement, we did not detect tPA in day 3 differentiating adipocytes, while a significant increase of tPA levels was observed in day 3 osteoblasts as compared to day 0 (Fig. 5C). By contrast, uPA remained undetectable in the secretion media of hMADS cells under the three conditions analyzed.

Bottom Line: Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly.Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2.Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome.

View Article: PubMed Central - HTML - PubMed

Affiliation: ISBDC, Université de Nice Sophia-Antipolis, CNRS ; 28 avenue de Valrose, 06100 Nice, France. Chiara.Chiellini@gmail.com

ABSTRACT

Background: It is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS) at an early step of commitment to adipocytes and osteoblasts.

Results: A proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 73 proteins at day 0 and day 3 of adipocyte and osteoblast differentiation. Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly. These proteins were classified into 8 clusters according to their function. Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2. Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome. Furthermore we showed that PAI-1 mRNA was down-regulated in the bone of ovariectomized mice.

Conclusion: Given its regulation during the early events of hMADS cell differentiation and its status in ovariectomized mice, PAI-1 could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis.

Show MeSH
Related in: MedlinePlus