Genetic and functional modulation by agonist MRS5698 and allosteric enhancer LUF6000 at the native A3 adenosine receptor in HL-60 cells

Abstract

The A₃ adenosine receptor (AR) is an important inflammatory and immunological target. However, the underlying mechanisms are not fully understood. Here, we report the gene regulation in HL-60 cells treated acutely with highly selective A₃AR agonist MRS5698, positive allosteric modulator (PAM) LUF6000, or both. Both pro- and anti-inflammatory genes, such as IL-1a, IL-1β, and NFκBIZ, are significantly upregulated. During our observations, LUF6000 alone produced a lesser effect, while the MRS5698 + LUF6000 group demonstrated generally greater effects than MRS5698 alone, consistent with allosteric enhancement. The number of genes up- and down-regulated are similar. Pathway analysis highlighted the critical involvement of signaling molecules, including IL-6 and IL-17. Important upstream regulators include IL-1a, IL-1β, TNF-α, NF-κB, etc. PPAR, which modulates eicosanoid metabolism, was highly downregulated by the A₃AR agonist. Considering previous pharmacological results and mathematical modeling, LUF6000's small enhancement of genetic upregulation suggested that MRS5698 is a nearly full agonist, which we demonstrated in both cAMP and calcium assays. The smaller effect of LUF6000 on MRS5698 in comparison to its effect on Cl-IB-MECA was shown in both HL-60 cells endogenously expressing the human (h) A₃AR and in recombinant hA₃AR-expressing CHO cells, consistent with its HL-60 cell genetic regulation patterns. In summary, by using both selective agonists and PAM, we identified genes that are closely relevant to immunity and inflammation to be regulated by A₃AR in differentiated HL-60 cells, a cell model of neutrophil function. In addition, we demonstrated the previously uncharacterized allosteric signaling-enhancing effect of LUF6000 in cells endogenously expressing the hA₃AR.

Keywords: Adenosine receptor; Agonist; HL-60 cells; Microarray; Positive allosteric modulator.