“Tasting the Microenvironment”: Human Bone Marrow Stromal Cells Have Microenvironment Chemosensory Capacity Via Taste Receptor Expression

Abstract 938

The ability of cells to detect molecules in the microenvironment is important in cell signaling and cell responsiveness to environmental changes. Through an isobaric tag for relative and absolute quantitation (iTRAQ) based proteomic screen of human bone marrow stroma-derived cells (MSC) at the third passage we identified the novel expression of a bitter taste receptor, TAS2R46 in undifferentiated MSC and MSC differentiated into osteocytes, adipocytes, and cartilage. The relative amount of the receptor by iTRAQ and qRT-PCR was equivalent in all cell types. TAS2R46 expression was verified by flow cytometry, immunohistochemistry, and RT-PCR. Expression of TAS2R46 was also found in freshly obtained bone marrow mononuclear cells, and sorting by flow cytometry showed that the taste receptor positive cells had the ability to become MSC in vitro, while significantly fewer cells became MSC in the taste receptor negative/low fraction. Other members of the bitter taste receptor family including TAS2R4, TAS2R5, TAS2R1, TAS2R38 were negative by flow cytometry and iTRAQ. Bitter compounds, of which the prototypical molecule is denatonium, were found to increase intracellular human MSC calcium levels in a dose-responsive manner up to a level of 200% above baseline in fluorescent intracellular calcium detection assays. Other bitter compounds tested were caffeine, thujone, and salicin, and quinine gave rise to increased intracellular calcium levels. The specificity of the calcium response was verified through transgenic overexpression experiments, antibody inhibition, and a novel newly developed direct labeling method in which we were to directly label denatonium and show binding to the cell surface. Evaluation of downstream signaling events showed that interaction with this receptor caused a decrease in cAMP levels of 40% with exposure to 3 mM denatonium. Finally, to reveal the potential that this receptor may have, a native physiologically relevant function related to small peptide binding, a casein hydrosylate was used as a substrate. Remarkably, casein hydrosylate caused a similar increase in intracellular calcium as denatonium. Furthermore, this effect was augmented when TAS2R46 was overexpressed as a transgene. Current studies are working towards revealing the specific peptide that binds to this receptor. This is the first description of chemosensory detection by MSC. Our data show that this environmental detection occurs through a novel expression of a bitter taste receptor, TAS2R46. This ability may allow MSC to sample changes in their microenvironment triggered by small molecules, which could include either toxins or hormones as true natural ligands of this receptor class. The expression of bitter taste receptors may influence the response of MSCs to noxious materials or pathological insults to the organism that are detected as bitter substrates. Future studies will examine the directed mobility of MSCs to such stimuli which may offer new insights as to how MSCs sense organism injury.