Estrogen Up-regulates Neuropeptide Y Y1 Receptor Expression in a Human Breast Cancer Cell Line

Abstract

Normal breast tissue mainly expresses the neuropeptide Y (NPY) Y2 receptor whereas primary human breast carcinomas express the Y1 receptor (Y1R) subtype. We hypothesized that activation of estrogen signaling systems plays a role in the induction of Y1R. To investigate this possibility, we used estrogen receptor-positive (ER+) human breast carcinoma cell line, MCF-7, and examined the effect of estrogen on Y1R gene expression and its signaling pathways. Saturation binding studies revealed that MCF-7 cells express high-affinity NPY receptor. NPY inhibited forskolin-stimulated adenosine 3V 5V -cyclic monophosphate (cAMP) accumulation and mobilized intracellular Ca 2+ in MCF-7 cells. Chronic estrogen treatment enhanced NPY-mediated inhibition of cAMP accumulation by 4-fold and caused a significant increase in Y1R mRNA expression through ERA. Similarly, estrogen increased Y1R mRNA expression in T-47D (ER+) but not in MDA-MB231 or MDA-MB468 (ERÀ) cell lines. Cycloheximide decreased basal Y1R mRNA expression; however, it did not affect its increase by estrogen. Moreover, estrogen treatment of MCF-7 cells did not increase Y1R mRNA stability. The up-regulation of Y1R expression by estrogen is prevented by hydroxyurea but not by nocodazole or IB-MECA (cell cycle inhibitors). Lastly, NPY inhibited estrogen-induced cell proliferation through Y1R. In conclusion, MCF-7 cells express a functional Y1R coupled to both Ca 2+ and cAMP pathways. Estrogen up-regulates Y1R expression through ERA. This effect is independent of increased Y1R mRNA stability or new protein synthesis, and likely occurs during S phase completion of the cell cycle. Estrogen plays an important role in the up-regulation of Y1R, which in turn regulates estrogen-induced cell proliferation in breast cancer cells. (Cancer Res 2006; 66(7): 3706-14)

Neuropeptide Y Y5 Receptor Promotes Cell Growth through Extracellular Signal-Regulated Kinase Signaling and Cyclic AMP Inhibition in a Human Breast Cancer Cell Line

“…Further, our studies showed that the activation of Y1R attenuates estrogen-induced cell proliferation in MCF-7 (17).…”

“…Our recent findings showed that human breast cancer cell line MCF-7 expresses functional Y1R, which mobilizes intracellular Ca 2+ and decreases cyclic AMP (cAMP) accumulation in response to NPY stimulation (17). Further, our studies showed that the activation of Y1R attenuates estrogen-induced cell proliferation in MCF-7 (17).…”

“…Membrane fractions were incubated in the binding buffer as described in our previous report (17). In brief, 50 to 75 μg membrane protein were incubated in a final volume of 0.…”

“…In brief, 50 to 75 μg membrane protein were incubated in a final volume of 0. 25 (17). In standardized competition binding studies, a time dependency was determined and 2-h incubation at room temperature was found to be optimal.…”

“…cAMP Accumulation Studies in BT-549 Cells cAMP accumulation was determined in BT-549 cells as we previously described for MCF-7 cells (17). In brief, cells were grown in 24-well plates for 2 to 3 d. At 80% confluency, cells were incubated with serum-free DMEM-F12 medium containing 0.1% BSA, and 5 mmol/L theophylline for 1 h at 37°C.…”