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Mesenchymal Stem Cells Exhibit Regulated Exocytosis in Response to Chemerin and IGF.

Kumar JD, Holmberg C, Balabanova S, Borysova L, Burdyga T, Beynon R, Dockray GJ, Varro A - PLoS ONE (2015)

Bottom Line: The calcium ionophore, ionomycin, also rapidly increased secretion of TGFβig-h3 while inhibitors of translation (cycloheximide) or secretory protein transport (brefeldin A) had no effect, indicating secretion from preformed secretory vesicles.Inhibitors of the chemerin and IGF receptors specifically reduced the secretory response.Gene ontology analysis of proteins exhibiting regulated secretion indicated functions primarily associated with cell adhesion and in bioassays chemerin increased adhesion of MSCs and adhesion, proliferation and migration of myofibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Crown St, Liverpool, United Kingdom.

ABSTRACT
Mesenchymal stem cells (MSCs) play important roles in tissue repair and cancer progression. Our recent work suggests that some mesenchymal cells, notably myofibroblasts exhibit regulated exocytosis resembling that seen in neuroendocrine cells. We now report that MSCs also exhibit regulated exocytosis. Both a G-protein coupled receptor agonist, chemerin, and a receptor tyrosine kinase stimulant, IGF-II, evoked rapid increases in secretion of a marker protein, TGFβig-h3. The calcium ionophore, ionomycin, also rapidly increased secretion of TGFβig-h3 while inhibitors of translation (cycloheximide) or secretory protein transport (brefeldin A) had no effect, indicating secretion from preformed secretory vesicles. Inhibitors of the chemerin and IGF receptors specifically reduced the secretory response. Confocal microscopy of MSCs loaded with Fluo-4 revealed chemerin and IGF-II triggered intracellular Ca2+ oscillations requiring extracellular calcium. Immunocytochemistry showed co-localisation of TGFβig-h3 and MMP-2 to secretory vesicles, and transmission electron-microscopy showed dense-core secretory vesicles in proximity to the Golgi apparatus. Proteomic studies on the MSC secretome identified 64 proteins including TGFβig-h3 and MMP-2 that exhibited increased secretion in response to IGF-II treatment for 30min and western blot of selected proteins confirmed these data. Gene ontology analysis of proteins exhibiting regulated secretion indicated functions primarily associated with cell adhesion and in bioassays chemerin increased adhesion of MSCs and adhesion, proliferation and migration of myofibroblasts. Thus, MSCs exhibit regulated exocytosis that is compatible with an early role in tissue remodelling.

No MeSH data available.


Related in: MedlinePlus

Western blot validation of proteomic studies of the MSC secretome.MSCs were treated with chemerin or IGF-II and media or cell extracts probed by western blot for proteins identified by SILAC. Six proteins that exhibited increased secreted in proteomic studies were also increased in western blots response to IGF (MMP-2, TGFβig-h3, MIF, IGFBP-7, decorin and lumican) and all except one (IGFBP-7) were also stimulated by chemerin. SPARC was not identified as exhibiting stimulated exocytosis in proteomic studies and neither did it respond to IGF-II or chemerin in western blot studies. Cellular content of SPARC and GAPDH was not influenced by IGF-II or chemerin.
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pone.0141331.g004: Western blot validation of proteomic studies of the MSC secretome.MSCs were treated with chemerin or IGF-II and media or cell extracts probed by western blot for proteins identified by SILAC. Six proteins that exhibited increased secreted in proteomic studies were also increased in western blots response to IGF (MMP-2, TGFβig-h3, MIF, IGFBP-7, decorin and lumican) and all except one (IGFBP-7) were also stimulated by chemerin. SPARC was not identified as exhibiting stimulated exocytosis in proteomic studies and neither did it respond to IGF-II or chemerin in western blot studies. Cellular content of SPARC and GAPDH was not influenced by IGF-II or chemerin.

Mentions: In order to validate the proteomic identification of the regulated MSC secretome, we performed western blot on a subset of secreted proteins selected on the basis of representation of different functional classes and including an example of a putative constitutively secreted protein (SPARC). All of 6 proteins (TGFβig-h3, MMP-2, MIF, decorin, lumican, IGFBP-7) exhibiting an increased relative abundance after IGF-II treatment in SILAC studies were also increased in western analysis (Fig 4), although curiously, one (IGFBP-7) exhibited a response to IGF-II but not chemerin. In contrast, SPARC was not stimulated in either the SILAC analysis or in western blots (Fig 4). The secretion of selected proteins exhibiting IGF-II or chemerin-stimulated exocytosis was rapid with detectable responses after just 5 min of stimulation (S4 Fig), was resistant to BFA treatment (S5 Fig) but was sensitive to AG1024 or CCX832, respectively (S6 Fig).


Mesenchymal Stem Cells Exhibit Regulated Exocytosis in Response to Chemerin and IGF.

Kumar JD, Holmberg C, Balabanova S, Borysova L, Burdyga T, Beynon R, Dockray GJ, Varro A - PLoS ONE (2015)

Western blot validation of proteomic studies of the MSC secretome.MSCs were treated with chemerin or IGF-II and media or cell extracts probed by western blot for proteins identified by SILAC. Six proteins that exhibited increased secreted in proteomic studies were also increased in western blots response to IGF (MMP-2, TGFβig-h3, MIF, IGFBP-7, decorin and lumican) and all except one (IGFBP-7) were also stimulated by chemerin. SPARC was not identified as exhibiting stimulated exocytosis in proteomic studies and neither did it respond to IGF-II or chemerin in western blot studies. Cellular content of SPARC and GAPDH was not influenced by IGF-II or chemerin.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4626093&req=5

pone.0141331.g004: Western blot validation of proteomic studies of the MSC secretome.MSCs were treated with chemerin or IGF-II and media or cell extracts probed by western blot for proteins identified by SILAC. Six proteins that exhibited increased secreted in proteomic studies were also increased in western blots response to IGF (MMP-2, TGFβig-h3, MIF, IGFBP-7, decorin and lumican) and all except one (IGFBP-7) were also stimulated by chemerin. SPARC was not identified as exhibiting stimulated exocytosis in proteomic studies and neither did it respond to IGF-II or chemerin in western blot studies. Cellular content of SPARC and GAPDH was not influenced by IGF-II or chemerin.
Mentions: In order to validate the proteomic identification of the regulated MSC secretome, we performed western blot on a subset of secreted proteins selected on the basis of representation of different functional classes and including an example of a putative constitutively secreted protein (SPARC). All of 6 proteins (TGFβig-h3, MMP-2, MIF, decorin, lumican, IGFBP-7) exhibiting an increased relative abundance after IGF-II treatment in SILAC studies were also increased in western analysis (Fig 4), although curiously, one (IGFBP-7) exhibited a response to IGF-II but not chemerin. In contrast, SPARC was not stimulated in either the SILAC analysis or in western blots (Fig 4). The secretion of selected proteins exhibiting IGF-II or chemerin-stimulated exocytosis was rapid with detectable responses after just 5 min of stimulation (S4 Fig), was resistant to BFA treatment (S5 Fig) but was sensitive to AG1024 or CCX832, respectively (S6 Fig).

Bottom Line: The calcium ionophore, ionomycin, also rapidly increased secretion of TGFβig-h3 while inhibitors of translation (cycloheximide) or secretory protein transport (brefeldin A) had no effect, indicating secretion from preformed secretory vesicles.Inhibitors of the chemerin and IGF receptors specifically reduced the secretory response.Gene ontology analysis of proteins exhibiting regulated secretion indicated functions primarily associated with cell adhesion and in bioassays chemerin increased adhesion of MSCs and adhesion, proliferation and migration of myofibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Crown St, Liverpool, United Kingdom.

ABSTRACT
Mesenchymal stem cells (MSCs) play important roles in tissue repair and cancer progression. Our recent work suggests that some mesenchymal cells, notably myofibroblasts exhibit regulated exocytosis resembling that seen in neuroendocrine cells. We now report that MSCs also exhibit regulated exocytosis. Both a G-protein coupled receptor agonist, chemerin, and a receptor tyrosine kinase stimulant, IGF-II, evoked rapid increases in secretion of a marker protein, TGFβig-h3. The calcium ionophore, ionomycin, also rapidly increased secretion of TGFβig-h3 while inhibitors of translation (cycloheximide) or secretory protein transport (brefeldin A) had no effect, indicating secretion from preformed secretory vesicles. Inhibitors of the chemerin and IGF receptors specifically reduced the secretory response. Confocal microscopy of MSCs loaded with Fluo-4 revealed chemerin and IGF-II triggered intracellular Ca2+ oscillations requiring extracellular calcium. Immunocytochemistry showed co-localisation of TGFβig-h3 and MMP-2 to secretory vesicles, and transmission electron-microscopy showed dense-core secretory vesicles in proximity to the Golgi apparatus. Proteomic studies on the MSC secretome identified 64 proteins including TGFβig-h3 and MMP-2 that exhibited increased secretion in response to IGF-II treatment for 30min and western blot of selected proteins confirmed these data. Gene ontology analysis of proteins exhibiting regulated secretion indicated functions primarily associated with cell adhesion and in bioassays chemerin increased adhesion of MSCs and adhesion, proliferation and migration of myofibroblasts. Thus, MSCs exhibit regulated exocytosis that is compatible with an early role in tissue remodelling.

No MeSH data available.


Related in: MedlinePlus