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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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Related in: MedlinePlus

Secretion levels of 8 candidates released from hMADS cells during the commitment to adipocytes and osteoblasts. The activity of MMP2 has been evaluated by gelatin zymography (A). The expression of SPARC (B), ENO1(C), PEDF (D), GRP78 (E), BIGH3 (F), PTX3 (G) and PAI-1 (H) has been analyzed by Western blot. The bar graphs report the levels of expression of every single candidate as the mean of three independent experiments after 6 h of incubation. The values are indicated as arbitrary units. *: p < 0.05. Two μg of secreted proteins have been loaded for each gel.
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Figure 4: Secretion levels of 8 candidates released from hMADS cells during the commitment to adipocytes and osteoblasts. The activity of MMP2 has been evaluated by gelatin zymography (A). The expression of SPARC (B), ENO1(C), PEDF (D), GRP78 (E), BIGH3 (F), PTX3 (G) and PAI-1 (H) has been analyzed by Western blot. The bar graphs report the levels of expression of every single candidate as the mean of three independent experiments after 6 h of incubation. The values are indicated as arbitrary units. *: p < 0.05. Two μg of secreted proteins have been loaded for each gel.

Mentions: Mass spectrometry identification data were validated for 8 candidates, selecting them from different clusters, i.e. 1 protease, 2 protease inhibitors, 2 ECM components, 1 anti-inflammatory/anti-oxidant protein, 1 metabolic enzyme and 1 heat shock protein. Western blots or zymograms were employed to supplement the identification by mass spectrometry and to determine the expression profile among three different sets of conditions (day 0 and day 3 for adipogenesis or osteogenesis). For all the candidates, the results of immunoblotting/zymogram and a semi-quantification of the bands are reported in Figure 4.


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)

Secretion levels of 8 candidates released from hMADS cells during the commitment to adipocytes and osteoblasts. The activity of MMP2 has been evaluated by gelatin zymography (A). The expression of SPARC (B), ENO1(C), PEDF (D), GRP78 (E), BIGH3 (F), PTX3 (G) and PAI-1 (H) has been analyzed by Western blot. The bar graphs report the levels of expression of every single candidate as the mean of three independent experiments after 6 h of incubation. The values are indicated as arbitrary units. *: p < 0.05. Two μg of secreted proteins have been loaded for each gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Secretion levels of 8 candidates released from hMADS cells during the commitment to adipocytes and osteoblasts. The activity of MMP2 has been evaluated by gelatin zymography (A). The expression of SPARC (B), ENO1(C), PEDF (D), GRP78 (E), BIGH3 (F), PTX3 (G) and PAI-1 (H) has been analyzed by Western blot. The bar graphs report the levels of expression of every single candidate as the mean of three independent experiments after 6 h of incubation. The values are indicated as arbitrary units. *: p < 0.05. Two μg of secreted proteins have been loaded for each gel.
Mentions: Mass spectrometry identification data were validated for 8 candidates, selecting them from different clusters, i.e. 1 protease, 2 protease inhibitors, 2 ECM components, 1 anti-inflammatory/anti-oxidant protein, 1 metabolic enzyme and 1 heat shock protein. Western blots or zymograms were employed to supplement the identification by mass spectrometry and to determine the expression profile among three different sets of conditions (day 0 and day 3 for adipogenesis or osteogenesis). For all the candidates, the results of immunoblotting/zymogram and a semi-quantification of the bands are reported in Figure 4.

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