Deorphanization and characterization of the ectopically expressed olfactory receptor OR51B5 in myelogenous leukemia cells

Abstract

The ectopic expression of olfactory receptors (ORs) in the human body has been of major interest in the past decade. Several studies have reported the expression of ORs not only in healthy tissues such as heart, sperm or skin cells, but also in cancerous tissues of the liver, prostate or intestine. In the present study, we detected the expression of OR51B5 in the chronic myelogenous leukemia (CML) cell line K562 and in white blood cell samples of clinically diagnosed acute myelogenous leukemia (AML) patients by reverse transcription-PCR and immunocytochemical staining. The known OR51B5 ligand isononyl alcohol increased the levels of intracellular Ca(2+) in both AML patient blood cells and K562 cells. With calcium imaging experiments, we characterized in greater detail the OR51B5-mediated signaling pathway. Here, we observed an involvement of adenylate cyclase and the downstream L-type and T-type calcium channels. In addition, the activation of OR51B5 leads to an inhibition of cell proliferation in K562 cells. In western blot experiments, we found that incubation with isononyl alcohol led to a reduction in p38-MAPK (mitogen-activated protein kinase) phosphorylation that might be responsible for the decreased cell proliferation. In the present study, we characterized the OR51B5-mediated signaling pathway downstream of the activation with isononyl alcohol, which leads to reduced proliferation and therefore provide a novel pharmacological target for CML and AML, the latter of which remains difficult to treat.

Figure 1

OR51B5 is expressed in the CML cell line K562. (a) RT-PCR experiments show the expression of OR51B5 in K562 cells at the expected size of ~230 bp. GAPDH was used as a control. (b) qPCR experiments showed that after 4 h isononyl alcohol incubation OR51B5 expression was significantly decreased. The effect was enhanced by longer stimulation time (24 h). (c) Immunocytochemical staining with the OR51B5 antibody validates the expression of OR51B5 in K562 cells. DAPI was used to stain the cell nuclei.

Figure 2

Calcium imaging experiments with the CML cell line K562. (a) Application of different concentrations of isononyl alcohol increased the levels of intracellular Ca²⁺. (b) The EC₅₀ of isononyl alcohol in K562 cells was 3.4±0.2 mM (n=16). (c) Receptor desensitization was observed after repetitive isononyl alcohol stimulation. (d) Summarized results for the receptor desensitization after the application of different isononyl alcohol concentrations. (e) Long-term stimulation (6 min) with isononyl alcohol increased the cytosolic Ca²⁺ level within the first 2 min of the application time. ADP was used as a positive control to measure cell viability. (f) After 2 min of odor application, a maximum of ~58% of the cells showed an increase in their intracellular calcium levels.

Figure 3

Characterization of the OR51B5-mediated intracellular signaling components. (a) Under Ca²⁺-free conditions, an increase in cytosolic Ca²⁺ levels during isononyl alcohol stimulation was almost completely abolished. (b) During the co-treatment with isononyl alcohol and the AC inhibitor SQ-22536, no Ca²⁺ signals could be detected. (c) The PKA inhibitor H-89 blocked the isononyl alcohol-induced increase in intracellular Ca²⁺. (d) The PLC inhibitor U73122 could not significantly block the isononyl alcohol-mediated Ca²⁺ increase. (e) L-type calcium channel inhibitor Verapamil abolished the isononyl alcohol-induced Ca²⁺ increase in K562 cells. (f) The inhibitor experiments were analyzed relative to the first and second applications of isononyl alcohol.

Figure 4

Calcium imaging experiments in the native blood samples of clinically diagnosed AML patients. (a) Native white blood cells from AML patients showed an increase in their intracellular Ca²⁺ levels during isononyl alcohol application. (b) Representative calcium imaging with the application of 100 μM isononyl alcohol. (c) SQ-22536 abolished the isononyl alcohol-induced increase in intracellular Ca²⁺. (d) In absence of extracellular Ca²⁺ the isononyl alcohol-induced increase in intracellular Ca²⁺ was abolished. (e) Isononyl alcohol (100 μM) led to a significant increase in intracellular Ca²⁺ in ~35% of all AML cells. SQ-22536 and the extracellular Ca²⁺ chelator EGTA reduced the number of responding cells.

Figure 5

Examination of the protein kinase phosphorylation after isononyl alcohol application. (a) Exemplary western blots are shown for the alterations in the phosphorylation of protein kinases during isononyl alcohol incubation. Vinculin was used as a loading control. (b) Summarized results for the phosphorylation of various protein kinases. After 60 min of isononyl alcohol incubation, only p38-MAPK phosphorylation was significantly reduced.

Figure 6

Isononyl alcohol decreases the proliferation of K562 cells. (a) Isononyl alcohol (1 mM) significantly decreased cell proliferation within 24 h. After 5 days the cell proliferation was reduced up to ~25% compared with the control cells. (b) Control cells (Ctrl: DMSO 0.1%) did not exhibit altered cell proliferation compared with cells cultured in DMSO-free RPMI medium. Isononyl alcohol is able to decrease cell proliferation in time- and concentration-dependent manners. The strongest effect was observed after 5 days of incubation with 1 mM isononyl alcohol.