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

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.

Fig 1. Secretion of TGFβig-h3 is stimulated by chemerin and IGF.

A. Western blot analysis of TGFβig-h3 in media from MSC cells shows stimulation by chemerin and IGF-II and inhibition by CCX832 and AG1042, respectively. B. Stimulated secretion of TGFβig-h3 is maintained after cycloheximide treatment. TGFβig-h3 abundance in cell extracts was unchanged (middle panel); GAPDH was used as a loading control for the cell extracts (bottom panel). C. The calcium ionophore, ionomycin (1μM) stimulated TGFβig-h3 secretion comparable to IGF-I (50ng.ml^-1) and IGF-II (100ng.ml^-1). D. In calcium-free medium stimulated secretion in response to IGF-II is inhibited.

Fig 2. Effects of IGF and chemerin on Ca²⁺ signalling in MSCs.

A. Images of Fluo-4 loaded MSCs taken in the absence (left) and the presence (right) of chemerin (100nM), respectively. B. IGF-II (100 ng.ml^-1, top) and chemerin (100nM, Ch) induce Ca²⁺ oscillations in Fluo-4 loaded cells. C. Relaxation of Ca²⁺ transients induced by chemerin or IGF-II after removal of external Ca²⁺ (Ca²⁺ free solution with 2mM EGTA).

Fig 3. Enriched molecular functions, protein classes and biological processes in the MSC secretome.

A, The main molecular functions shown by PANTHER for 62 proteins in the MSC secretome identified as “secreted” and as exhibiting increased abundance after IGF-II treatment. B. Enriched protein classes, C. Enriched biological processes. P, probability.

Fig 4. 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.

Fig 5. Localisation of proteins in secretory vesicles.

A. localisation of TGFβig-h3 (green) and MMP2, MIF or SPACR (red) in MSCs revealed by immunocytochemistry. In each case there are images for the red channel, green channel, a marge of these two images, and finally a merged image at higher magnification. There are clear examples of TGFβig-h3 and either MIF or MMP-2 in similar vesicular populations (yellow or orange), while TGFβig-h3 and SPARC are localised to distinct vesicular structures (red or green vesicular structures). Nuclei stained blue with DAPI. Scale bars 5 or 10 μm. B. TEM identifies dense core secretory vesicles in MSCs. representative photomicrograph taken at a magnification of 60,000x show dense core secretory vesicles (closed arrows) in MSCs the Golgi apparatus is clearly visible (open arrows). Scale bar 500nm.

Fig 6. Chemerin-conditioned medium increases cell adhesion, proliferation and migration.

A. Increased adhesion of MSCs (1x10⁵) after treatment with chemerin (Ch) for 30 min. B. Conditioned medium (CM) from MSCs treated with chemerin increased adhesion of normal gastric myofibroblasts and addition of the ChemR23 antagonist CCX832 only slightly reduced the response. C. CM from MSCs treated with chemerin stimulated migration of myofibroblasts in Boyden chambers and addition of the ChemR23 antagonist CCX832 only slightly reduced the response. D. CM from MSCs treated with chemerin stimulated proliferation of myofibroblasts and addition of the ChemR23 antagonist CCX832 only slightly reduced the response. Means ± SE, n = 3; horizontal arrows, p<0.05.