Contributions
Abstract: EP753
Type: E-Poster Presentation
Session title: Hematopoiesis, stem cells and microenvironment
Background
The ability of cells to detect changes in the microenvironment is important in cell signaling and responsiveness to environmental fluctuations. There are a variety of receptor types through which cells respond to changes in their microenvironment including those for cytokines, hormones, mechanical tension, and there are thousands of different receptors expressed on any given cell type. Among these, the largest group belongs to the family of G protein-couple receptors (GPCRs). Bitter taste receptors (T2Rs) comprise 25 distinct members of the GPCR family. Initially described in the oral cavity, T2Rs are actually widely expressed in different tissues and the increasing amount of evidence about their extra-oral localization has suggested a wider function in sensing microenvironment, also in cancer settings. We recently reported that Acute Myeloid Leukemia cells express fully functional T2R subtypes which are involved in the regulation of leukemia cell functions.
Aims
In this study, for the first time, we investigate the activity of bitter taste agonist Denatonium on HSCs biology.
Methods
Highly purified cord blood-derived CD34+ cells were used for this study. T2R expression was analyzed by RealTimePCR and IF. We investigated molecular profiling by GEP assay, viability by colorimetric assay, apoptosis and HSC subset frequency by FACS analysis. In vitro assays for primitive hematopoietic cells (CFU-C, LTC-IC) were performed. For in vivo engraftment studies, NSG mice were used.
Results
In the present work, we show that HSCs expressed several T2R subtypes at molecular and protein levels. Stimulation of HSCs with a T2R agonist, Denatonium (DEN), induced intracellular Ca2+ concentration increase, thus demonstrating T2Rs functionality. GEP analysis identified a number of genes significantly modulated by DEN treatment. Specifically, genes involved in the stem cell population maintenance and regulation of cell-matrix adhesion are deregulated. Functional assays demonstrated that DEN exposure does not affect HSC viability and not induce apoptosis. Nevertheless, depending on the extent of stimulation, DEN modulates the frequency of stem cell progenitors in the culture. In particular, acute stimulation increases the frequency of lineage restricted progenitors (CLP, CMP, GMP) and their clonogenic capacity. On the other hand, chronic exposure to DEN supported the depletion of the more primitive stem cell compartment, reducing the frequency of the LTC-IC. These results are supported by in vivo experiments. NSG mice transplanted with DEN-treated HSC showed an increased engraftment in BM and spleen matched by an increased frequency of more differentiated progenitors.
Conclusion
Our results expand the observation of T2R expression to the hematological setting and shed light on a role of T2Rs in the extrinsic regulation of HSC functions. Moreover, our data may suggest a role for microenvironment “bitter” molecules in regulating normal hematopoiesis. Notably, many common drugs, such as antibiotics, chloroquine, haloperidol, procainamide, are bitter tasting and are thus effective ligands for T2Rs. For this reason, they could exhibit off-target effect in T2R expressing cells, including normal hematopoietic cells. Our results may have implications in understanding the off-target actions of diverse drugs.
Keyword(s): Hematopoietic stem and progenitor cells
Abstract: EP753
Type: E-Poster Presentation
Session title: Hematopoiesis, stem cells and microenvironment
Background
The ability of cells to detect changes in the microenvironment is important in cell signaling and responsiveness to environmental fluctuations. There are a variety of receptor types through which cells respond to changes in their microenvironment including those for cytokines, hormones, mechanical tension, and there are thousands of different receptors expressed on any given cell type. Among these, the largest group belongs to the family of G protein-couple receptors (GPCRs). Bitter taste receptors (T2Rs) comprise 25 distinct members of the GPCR family. Initially described in the oral cavity, T2Rs are actually widely expressed in different tissues and the increasing amount of evidence about their extra-oral localization has suggested a wider function in sensing microenvironment, also in cancer settings. We recently reported that Acute Myeloid Leukemia cells express fully functional T2R subtypes which are involved in the regulation of leukemia cell functions.
Aims
In this study, for the first time, we investigate the activity of bitter taste agonist Denatonium on HSCs biology.
Methods
Highly purified cord blood-derived CD34+ cells were used for this study. T2R expression was analyzed by RealTimePCR and IF. We investigated molecular profiling by GEP assay, viability by colorimetric assay, apoptosis and HSC subset frequency by FACS analysis. In vitro assays for primitive hematopoietic cells (CFU-C, LTC-IC) were performed. For in vivo engraftment studies, NSG mice were used.
Results
In the present work, we show that HSCs expressed several T2R subtypes at molecular and protein levels. Stimulation of HSCs with a T2R agonist, Denatonium (DEN), induced intracellular Ca2+ concentration increase, thus demonstrating T2Rs functionality. GEP analysis identified a number of genes significantly modulated by DEN treatment. Specifically, genes involved in the stem cell population maintenance and regulation of cell-matrix adhesion are deregulated. Functional assays demonstrated that DEN exposure does not affect HSC viability and not induce apoptosis. Nevertheless, depending on the extent of stimulation, DEN modulates the frequency of stem cell progenitors in the culture. In particular, acute stimulation increases the frequency of lineage restricted progenitors (CLP, CMP, GMP) and their clonogenic capacity. On the other hand, chronic exposure to DEN supported the depletion of the more primitive stem cell compartment, reducing the frequency of the LTC-IC. These results are supported by in vivo experiments. NSG mice transplanted with DEN-treated HSC showed an increased engraftment in BM and spleen matched by an increased frequency of more differentiated progenitors.
Conclusion
Our results expand the observation of T2R expression to the hematological setting and shed light on a role of T2Rs in the extrinsic regulation of HSC functions. Moreover, our data may suggest a role for microenvironment “bitter” molecules in regulating normal hematopoiesis. Notably, many common drugs, such as antibiotics, chloroquine, haloperidol, procainamide, are bitter tasting and are thus effective ligands for T2Rs. For this reason, they could exhibit off-target effect in T2R expressing cells, including normal hematopoietic cells. Our results may have implications in understanding the off-target actions of diverse drugs.
Keyword(s): Hematopoietic stem and progenitor cells