T cell chemotaxis to lysophosphatidylcholine through the G2A receptor

Abstract

G2A is an immunoregulatory G protein-coupled receptor predominantly expressed in lymphocytes and macrophages. Ectopic overexpression studies have implicated G2A as a receptor for the bioactive lysophospholipid, lysophosphatidylcholine (LPC). However, the functional consequences of LPC-G2A interaction at physiological levels of receptor expression, and in a cellular context relevant to its immunological role, remain largely unknown. Here, we show impaired chemotaxis to LPC of a T lymphoid cell line in which G2A expression was chronically down-regulated by RNA interference technology. Rescuing this phenotype by reconstitution of the physiological level of receptor expression further supports a functional connection between LPC-G2A interaction and cellular motility. Overexpression of G2A in the T lymphoid cell line significantly enhanced chemotaxis to LPC. It also modified migration toward the LPC-related molecule, lysophosphatidic acid, indicating the possibility of crosstalk between G2A and endogenous lysophosphatidic acid receptors. The role of G2A in LPC-mediated cell migration may be relevant to the autoimmune syndrome associated with genetic inactivation of this G protein-coupled receptor in mice. The experimental system described here can be useful for understanding the structural requirements for LPC recognition by G2A and the signaling pathways regulated by this ligand-receptor pair.

Fig. 1.

Expression of G2A in DO11.10 cells. (A) (Upper) Detection of G2A by Western blot by using the rabbit polyclonal antibody. (Lower) Extracellular signal-regulated kinase 2 loading control using a polyclonal rabbit antibody from Santa Cruz Biotechnology. (B)(Upper) RT-PCR analysis of the expression of LP-specific GPCRs and of ATX in DO11.10 cells. (Lower) Control RT-PCR by using a mixture of cDNAs from mouse brain, heart, spleen, and prostate.

Fig. 2.

Cotransfection of EGFP-tagged GPCRs and sh RNAs encoding plasmids to select siRNA target sequences. HEK 293T cells were transfected with expression vectors encoding G2A-EGFP (A) or TDAG8-EGFP (B) fusions, alone or in combination shRNA plasmids. Forty-eight hours later, the level of EGFP fluorescence was determined by fluorescence-activated cell sorting and used as an indicator of silencing efficiency.

Fig. 3.

siRNA-mediated silencing of G2A in DO11.10 cells. (A) Retroviral vector design. The bidirectional human H1-RNA promoter (H1) coordinates expression of the effector shRNA (RNA polymerase III-dependent) and of EGFP (RNA polymerase II-dependent). Reverse transcription results in the duplication of the shRNA cassette inserted in the 3′ self-inactivating retroviral LTR (3′ SIN LTR): N21, target sequence, sense; RN21, target sequence, antisense; (T)5, termination signal for the RNA polymerase III; pA, polyadenylation signal. (B) Expression of the EGFP-colinked marker by retrovirally transduced DO11.10 cells. (C)(Upper) Western blot analysis by using the rabbit polyclonal antibody of the expression of G2A in DO11.10 T cells transduced with G2A-specific (G2A^shRNA) or control (CTR^shRNA) shRNA encoding retroviruses. (Lower) Extracellular signal-regulated kinase 2 loading control.

Fig. 4.

Chronic siRNA-mediated suppression of G2A is well tolerated by DO11.10 cells. (A) DNA synthesis rate. The 5 × 10⁴ WT or G2A-silenced DO11.10 cells (G2A^shRNA) in SFM containing 0.1% FAF-BSA were treated with indicated concentrations of LPC. The rate of DNA synthesis was determined by pulsing the cells with [³H]thymidine for 12 h. (B) IL-2 production. The 5 × 10⁴ DO11.10 cells in SFM with 0.1% FAF-BSA were stimulated for 24 h with 5 μg/ml plate-bound antibody against the CD3ε chain (2C11-145) in the presence of indicated amounts of LPC. Production of IL-2 was determined by capture ELISA. The results are representative of three independent experiments. ^*, P > 0.05; ^**, P < 0.05. Student's t test was performed by using prism software (GraphPad, San Diego). P < 0.05 was considered to be statistically significant.

Fig. 5.

G2A-dependent chemotaxis to LPC. Equal numbers (2 × 10⁵) of WT and G2A^shRNA or control (CTR^shRNA) DO11.10 cells were washed three times with SFM containing 0.1% FAF-BSA, mixed, and added to the upper chamber of a 24-well plate with 5.0-μm pore size polycarbonate filters (Costar). LPC (A) or SDF1-α (B) was added to the lower chamber. After a 2-h incubation at 37°C in an 8% CO₂ incubator, transmigrated WT and EGFP-positive siRNA-transduced cells were counted by fluorescence-activated cell sorting. The results are representative of four independent experiments.

Fig. 6.

Reconstitution of G2A expression in shRNA-transduced cells rescues chemotaxis to LPC. (A) Schematic representation of the retroviral vector used for G2A reconstitution. The immediate early cytomegalovirus promoter (P_CMV IE) drives the expression of the puromycin resistance gene (puro^r). Expression of the siRNA refractory form of G2A is driven by the thymidine kinase promoter. (B) A Western blot to determine expression levels of G2A in WT, G2A^shRNA, and G2A^shRNA cells transduced with the G2A reconstitution vector (G2A^REC) and selected by using 1 μg/ml puromycin. (C) Chemotaxis of G2A^shRNA and G2A^REC cells to various concentrations of LPC. The results are representative of three independent experiments.

Fig. 7.

Effects of G2A overexpression in DO11.10 cells. (A) A Western blot to estimate the amount of G2A in cells overexpressing the receptor (G2A^HIGH, lysates from these cells were diluted 10- and 20-fold before analysis). Chemotaxis of WT and G2A^HIGH cells to LPC (B), S1P (C), and LPA (D) is shown. The results are representative of three independent experiments.