Full-length and truncated neurokinin-1 receptor expression and function during monocyte/macrophage differentiation

Abstract

The substance P (SP)-preferring receptor neurokinin-1 receptor (NK-1R) has two forms: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. These two receptors differ in the length of the C terminus of NK-1R. We studied the undifferentiated and phorbol myristate acetate (PMA)-differentiated human monocyte/macrophage cell line THP-1 to investigate the expression and function of NK-1R. The expression of full-length and truncated NK-1R in this cell line was determined by using real-time PCR and immunofluorescence staining. Undifferentiated THP-1 cells expressed only truncated NK-1R. The differentiation of THP-1 cells with PMA to a macrophage-like phenotype resulted in the expression of full-length NK-1R, which was functionally accompanied by an SP (10(-6) M)-induced Ca2+ increase. In contrast, the addition of SP (10(-6) M) did not trigger Ca2+ response in undifferentiated THP-1 cells; however, SP did enhance the CCR5-preferring ligand RANTES (CCL5)-mediated Ca2+ increase. When a plasmid containing the full-length NK-1R was introduced into undifferentiated THP-1 cells, exposure to SP triggered Ca2+ increase, demonstrating that the full-length NK-1R is required for SP-induced Ca2+ increase. The NK-1R antagonist aprepitant (Emend, Merck) inhibited both the SP-induced Ca2+ increase in PMA-differentiated THP-1 cells and the SP priming effect on the CCL5-mediated Ca2+ increase, indicating that these effects are mediated through the full-length and truncated NK-1R, respectively. Taken together, these observations demonstrate that there are unique characteristics of NK-1R expression and NK-1R-mediated signaling between undifferentiated THP-1 cells and THP-1 cells differentiated to the macrophage phenotype.

Fig. 1.

The effect of PMA differentiation of THP-1 cells on full-length and truncated NK-1R mRNA expression. (A) The effect of PMA differentiation of THP-1 on full-length NK-1R mRNA expression. ∗, The expression level of full-length NK-1R in day-12 post-PMA-treated THP-1 cells is significantly higher than that of undifferentiated (day 0; P < 0.01) and day-3 post-PMA differentiated cells (P < 0.05). (B) The effect of PMA differentiation of THP-1 cells on truncated NK-1R mRNA expression. The data represent the averages of three independent experiments.

Fig. 2.

Immunofluorescence staining of NK-1R in undifferentiated (A and B) and PMA-differentiated (C and D) THP-1 cells. Undifferentiated THP-1 cells stained positively only by anti-N-terminal antibody (B), but not by the anti-C-terminal antibody (A). PMA-differentiated THP-1 cells were stained positively by both anti-C-terminal (C) and anti-N-terminal (D) antibodies. (Magnification: ×400.) The data are representative of two independent experiments. Rabbit IgG was used as negative control (data not shown), which had the same background as the anti-C-terminal antibody-stained undifferentiated THP-1 cell.

Fig. 3.

Effect of SP on the calcium increase in undifferentiated (A) and PMA-differentiated (B) THP-1 cells. (A) Undifferentiated THP-1 cells were treated with SP (10⁻⁶ M) as indicated by the arrow. (B) Twelve-day-differentiated THP-1 cells were preincubated in the presence or absence of aprepitant (2 × 10⁻⁵ M) for 30 min, and then SP (10⁻⁶ M) was added, as indicated by the arrow. The data shown are representative of three independent experiments.

Fig. 4.

Expression of full-length NK-1R is required for an SP-induced calcium increase in undifferentiated THP-1 cells. SP (10⁻⁶ M) induced calcium responses in undifferentiated THP-1 cells (A), and a full-length NK-1R gene-containing plasmid transfected undifferentiated THP-1 cells (B). The data shown are representative of three independent experiments.

Fig. 5.

SP enhanced the CCL5-mediated calcium response via truncated NK-1R. Undifferentiated THP-1 cells were incubated in the presence or absence of aprepitant (2 × 10⁻⁵ M) for 30 min before the addition of buffer or SP (10 s), followed by CCL5 at 90 s. Addition of SP (10⁻⁶ M) at 10 s and CCL5 (10⁻⁸ M) at 100 s is indicated by arrows. The data shown are representative of three independent experiments.