Adenosine Signaling Regulates Adult Erythropoiesis.
Authors: Aerbajinai Wulin , Natarajan Bhanu V. , Gantt Nicole , Oneal Patricia , Goh Sung-Ho , Lee Y. Terry , Miller Jeffery L. 
Published: October 2019

Abstract
Adenosine signal transduction in human cells is achieved through four distinct G-protein-coupled receptors named A1, A2A, A2B and A3. Adenosine signaling events affect several physiological processes including growth regulation and cellular responses to hypoxia. Despite extensive studies of native and pharmacologically-targeted adenosine signal transduction, the potential for adenosine to regulate erythropoiesis remains largely unexplored. Here we systematically studied adenosine receptor expression, activation, and inhibition among CD34+ human erythroid progenitor cells cultured in the presence of erythropoietin. This 14 day culture system has been studied extensively in the context of erythroblast commitment and terminal differentiation (BLOOD 99(8):3005–13). Using real-time RT-PCR, we demonstrated that the A2A, A2B and A3 adenosine receptors are expressed in highly regulated patterns. The expression of A2A and A2B were highest early in the culture period and later declined as the cells underwent terminal differentiation. Receptor A3 gene expression gradually increased during the terminal differentiation. Expression of the A1 receptor gene was not detected above background levels at any stage of differentiation (compared with the positive control). Based upon those receptor expression patterns, several adenosine receptor signaling agonists and antagonists were screened for possible effects on erythroblast growth and differentiation. Among those screened molecules, the selective A3 adenosine receptor agonist, CI-IB-MECA, had effects on the proliferation and differentiation of the cells without overt toxicity in the dose range of 10–100uM. CI-IB-MECA significantly inhibited the proliferation of the cells with resulting cell counts being only 10% of those in matched controls after 14 days. Cell cycle analyses demonstrated that the growth inhibitory effects of CI-IB-MECA were largely due to a significant reduction in the percentage of S-phase proerythroblasts (S-phase: 16 ± 5% in CI-IB-MECA versus 51 ± 2% in matched controls; p<0.01). The level of apoptosis increased in CI-IB-MECA, but did not reach statistical significance (sub-G1 population: 8.3 ± 3% in CI-IB-MECA versus 1.5 ± 0.2% in matched controls; p= 0.08). Despite the profound inhibition of cell cycling, CI-IB-MECA did not prevent erythroblast commitment as evidenced by the expression of CD71 and Glycophorin A on the cell surface during the second culture week. However, CI-IB-MECA delayed the expression of those surface markers by 2–4 days when compared with the matched controls. The delayed maturation of the cells was also detected by morphological examination. On day 14, less than 10% of the cells were hemoglobinized erythroblasts in CI-IB-MECA compared with a predominance of those cells (>80%) in the matched controls. Consistent with the morphological examination, HPLC analyses revealed barely detectable levels of hemoglobin in the presence of CI-IB-MECA after 14 days. These finding suggest that selective activation of adenosine A3 receptors permits erythroid commitment, but profoundly inhibits the proliferation of those committed cells and retards their terminal differentiation. As a novel regulator of erythropoiesis, adenosine A3 receptor signaling should be explored in patients with growth-related erythroid diseases.