RasGRP1 is required for human NK cell function

Abstract

Cross-linking of NK activating receptors activates phospholipase-gamma and subsequently induces diacylglycerol and Ca(2+) as second messengers of signal transduction. Previous studies reported that Ras guanyl nucleotide-releasing protein (RasGRP) 1, which is activated by diacylglycerol and Ca(2+), is crucial for TCR-mediated Ras-ERK activation. We now report that RasGRP1, which can also be detected in human NK cells, plays an essential role in NK cell effector functions. To examine the role of RasGRP1 in NK cell functions, the expression of RasGRP1 was suppressed using RNA interference. Knockdown of RasGRP1 significantly blocked ITAM-dependent cytokine production as well as NK cytotoxicity. Biochemically, RasGRP1-knockdown NK cells showed markedly decreased ability to activate Ras, ERK, and JNK. Activation of the Ras-MAPK pathway was independently shown to be indispensable for NK cell effector functions via the use of specific pharmacological inhibitors. Our results reveal that RasGRP1 is required for the activation of the Ras-MAPK pathway leading to NK cell effector functions. Moreover, our data suggest that RasGRP1 might act as an important bridge between phospholipase-gamma activation and NK cell effector functions via the Ras-MAPK pathway.

Figure 1. RasGRP1 in NK cell cytotoxicity

(A) Expression of RasGRP1 in NK-92 and human primary NK cells. NK cell lysates were analyzed by western blotting with anti-RasGRP1 antibody. (B) Expression of RasGRP1 in NK cell precursors and mature NK cells. NK cell precursors were stimulated with IL-15 (30 ng/ml) for 0, 5, and 10 days, and mature NK cells were harvested. Expression of RasGRP1 was analyzed by western blotting. (C) Cell lysates of control shRNA- and RasGRP1 shRNA-expressing NK-92 cells were analyzed by western blotting with anti-RasGRP1 antibody. (D) Control shRNA- and RasGRP1 shRNA-expressing NK-92 cells were incubated with ⁵¹Cr-labeled K562 cells at the indicated effector/target (E:T) ratios. (E) Cell lysates of control siRNA- and RasGRP1 siRNA-nucleofected mature NK cells were analyzed by western blotting with anti-RasGRP1 antibody. (F) Control siRNA- and RasGRP1 siRNA-nucleofected mature NK cells were incubated with ⁵¹Cr-labeled K562 cells at the indicated E:T ratios. The data are representative of two independent experiments, and the error bars represent the standard deviation (s.d.) of triplicates.

Figure 2. Impaired ITAM-dependent cytokine production in RasGRP1-knockdown NK cells

(A) Control shRNA- and RasGRP1 shRNA-expressing NK-92 cells were stimulated with plate-bound antibodies to NKp30 (10 μg/ml), or were stimulated with PMA (1 ng/ml)/ionomycin (0.1 μg/ml) or IL-12 (20 ng/ml) in the culture medium. After 16 h, the cytokines (IFN-γ, TNF-α, and GM-CSF) released into the supernatant were measured by ELISA. * p < 0.05, ** p < 0.01. (B) shRNA-transduced NK-92 cells were stimulated with anti-NKp30 mAb (10 μg/ml), PMA (1 ng/ml)/ionomycin (0.1 μg/ml), or IL-12 (20 ng/ml) for 4 h, and the mRNA expression of the cytokines was analyzed by real-time PCR. * p < 0.05, ** p < 0.01. (C) Control siRNA- and RasGRP1 siRNA-nucleofected mature NK cells were stimulated with plate-bound antibodies to NCRs (10 μg/ml) or were stimulated with PMA (2 ng/ml)/ionomycin (0.2 μg/ml) or IL-12 (20 ng/ml) in the culture medium. After 16 h, the cytokines released into the supernatant were quantified by ELISA. * p < 0.05, ** p < 0.01. The data are representative of three independent experiments, and the error bars represent the s.d. of duplicates.

Figure 3. Reduced Ras activation in RasGRP1-knockdown NK cells

(A) Control shRNA- and RasGRP1 shRNA-expressing NK-92 cells were stimulated with plate-bound antibodies to NKp30 for the indicated times. Phosphorylation of PLC-γ1 and Src was detected by western blotting. (B) Control shRNA- and RasGRP1 shRNA-expressing NK-92 cells were pelleted with K562 cells or were stimulated with plate-bound antibodies to NKp30 for 5 min. Cell lysates were precipitated with GST-Raf-RBD, followed by western blotting with anti-pan-Ras antibody. (C) NK-92 cells were pretreated with FTI-277 (10 or 30 μM) for 30 min followed by incubation with ⁵¹Cr-labeled K562 cells at the indicated E:T ratios. (D) IFN-γ, TNF-α, and GM-CSF were measured by ELISA using the supernatant obtained from FTI-277 (10 or 30 μM)-pretreated NK-92 cells that were subsequently stimulated by NKp30 (10 μg/ml) cross-linking or PMA (1 ng/ml)/ionomycin (0.1 μg/ml). The data are representative of two independent experiments, and the error bars represent the s.d. of triplicates (C) or duplicates (D).

Figure 4. Impaired activation of ERK and JNK in RasGRP1-knockdown NK cells

Control shRNA- and RasGRP1 shRNA-expressing NK-92 cells (1×10⁶ cells/sample) were stimulated with (A) K562 (1×10⁵ cells/sample), (B) plate-bound antibodies to NKp30 (10 μg/ml), or (C) PMA (1 ng/ml)/ionomycin (0.1 μg/ml) for the indicated times. Phosphorylation of ERK, JNK and p38 was detected by western blotting. (D) Mature NK cells were pretreated with DMSO, the ERK inhibitor (PD98059; 10 μM), the JNK inhibitor (SP600125; 10 μM), or the p38 inhibitor (SB203580; 10 μM) for 30 min, followed by incubation with ⁵¹Cr-labeled K562 cells at the indicated E:T ratios. (E) Mature NK cells were treated with DMSO, PD98059 (10 μM), SP600125 (10 μM) or SB203580 (10 μM) for 30 min before stimulation. Pretreated mature NK cells were stimulated with plate-bound antibodies to NKp30 (10 μg/ml). Secreted IFN-γ was quantified by ELISA. The data are representative of three independent experiments, and the error bars represent the s.d. of triplicates (D) or duplicates (E).

Figure 5. Proposed model of RasGRP1 in NK cell receptor signaling

Upon cross-linking of NK activating receptors, PLC-γ is activated and induces DAG and IP₃-dependent Ca²⁺. DAG and Ca²⁺-bound RasGRP1 interacts with and activates Ras, which regulates activation of ERK and JNK. Finally, activated ERK and JNK evoke NK cytotoxicity and cytokine production.