The GTPase Rac regulates the proliferation and invasion of fibroblast-like synoviocytes from rheumatoid arthritis patients

Abstract

Fibroblast-like synoviocytes (FLS) isolated from joints of rheumatoid arthritis (RA) patients display proliferative and invasive properties reminiscent of those of malignant tumor cells. Rac small GTPases play an important role in tumor cell proliferation and invasion. We therefore investigated the potential role of Rac proteins in the proliferative and invasive behavior of RA-FLS. We showed that inhibiting Rac activity with the Rac-specific small molecule inhibitor NSC23766 causes a strong inhibition of RA-FLS proliferation, without affecting cell survival. Rac inhibition also results in a strong reduction in RA-FLS invasion through reconstituted extracellular matrix and a less marked inhibition of two-dimensional migration as measured by monolayer wound healing. We also showed that small interfering RNA-mediated depletion of Rac1 inhibits RA-FLS proliferation and invasion to a similar extent as NSC23766. These results demonstrate for the first time that Rac proteins play an important role in the aggressive behavior of FLS isolated from RA patients. In addition, we observed that inhibiting Rac proteins prevents JNK activation and that the JNK inhibitor SP600125 strongly inhibits RA-FLS invasion, suggesting that Rac-mediated JNK activation contributes to the role of Rac proteins in the invasive behavior of RA-FLS. In conclusion, Rac-controlled signaling pathways may present a new source of drug targets for therapeutic intervention in RA.

Figure 1

NSC23766 blocks Rac activity and RA-FLS proliferation. (A) RA-FLS were serum starved (control) in the presence or absence of 50 μM NSC23766 (NSC) for 24 h and subsequently stimulated with 10 ng/mL IL-1β for 5 min in the presence or absence of 50 μM NSC23766. Activated Rac (GTP-Rac) was extracted from cell lysates using GST-PAK and visualized by Western blotting using anti-Rac antibody. Total Rac from cell lysates before extraction was determined as loading control. Data shown are representative of two independent experiments. (B) RA-FLS (RA2) were grown overnight in 10 % serum in the absence (closed squares) or presence of 25 μM NSC23766 (open circles) or 50 μM NSC23766 (open squares). Subsequently, cells were plated on 96 well plates and cell growth was quantified using SRB staining. (C) Effect of NSC23766 on the proliferative activity of different RA-FLS cultures. Conditions as in (B). Solid bars: controls, empty bars: 50 μM NSC23766. Shown is the mean (± SEM) of five wells. For some of the data points, the error is smaller than the symbol size. * = P < 0.02, ** = P < 0.01 and *** = P < 0.001, two-tailed t test. Data for RA1 and RA2 are representative of respectively of three and two experiments. (D) Effect of NSC23766 on RA-FLS survival. RA-FLS (RA2) cells were treated with the indicated concentration of NSC23766 and apoptosis was measured using an ELISA assay that quantifies histone-associated DNA fragments. Shown are the mean values (± SEM) of three independent experiments from three RA-FLS cell lines.

Figure 2

Rac activity is necessary for RA-FLS invasion. Four different RA-FLS cultures were treated with 50 μM NSC23766 or control solution for 24 h. Cell invasion through Matrigel was quantified as described in Materials and Methods. Cell invasion numbers obtained with the different cultures were normalized to the respective controls. Shown is the mean (± SEM) of at least two independent experiments. ** = P < 0.01, two-tailed t test.

Figure 3

Decreased Rac activity inhibits cell migration. (A) Phase contrast micrographs of RA-FLS immediately and 21 h after wounding. RA-FLS (RA1) were pre-treated with 50 μM NSC23766 or control solution for 24 h and maintained in the presence or absence of 50 μM NSC23766 after wounding. Images of RA-FLS were taken at time 0 and 21 h after wounding. (B) Quantification of wound healing. RA-FLS (RA1) were treated with 50 μM NSC23766 (open squares) or control solution (closed squares) as described in (A). The migration distance was quantified as described in Materials and Methods. Shown are the mean values (± SEM) of eight measurements for each time point. Data are representative of two independent experiments. (C) Quantification of migration of control cells (solid bars) or cells treated with 50 μM NSC23766 (empty bars) after 21 h of various RA-FLS lines. *** = P < 0.001, two-tailed t test. Data shown are representative of two independent experiments.

Figure 4

Rac is necessary for lamellipodia formation in RA-FLS. RA-FLS (RA1) were plated overnight on coverslips and pre-treated for 1 h before wounding in the presence of 50 μM NSC23766, 20 μM SP600125 or DMSO. Cells were fixed and stained with fluorescent phalloidin 3 h after wounding to visualize polymerized actin in migrating cells. Data shown are representative of two independent experiments. Size bar represents 10 μm.

Figure 5

Inhibition of RA-FLS proliferation and invasion by siRNA-mediated depletion of Rac1. (A) Rac1 siRNA inhibits Rac expression. RA-FLS were transfected with Rac1 siRNA by nucleoporation. Rac protein levels were determined by Western blotting of cell lysates at the indicated days. Tubulin levels are shown as loading control. (B) Rac1-directed siRNA inhibits RA-FLS invasion. Matrigel invasion assays were performed on day seven post-transfection. Shown is the mean (± range) of two independent experiments. (C) Rac1-directed siRNA inhibits RA-FLS proliferation. SRB cell proliferation assays were initiated four days post-transfection. Shown is the mean (± range) of two independent experiments. *** = P < 0.001, two-tailed t test

Figure 6

Rac is necessary for JNK activation in RA-FLS. (A) RA-FLS (RA3) were serum starved (control) or treated with 50 μM NSC23766 for 24 h. JNK activity was determined by western blotting using phospho-JNK antibody, tubulin expression was determined as loading control. Similar results were obtained with the RA1 and RA2 cultures. (B) The JNK-specific inhibitor SP600125 inhibits RA-FLS invasion. RA-FLS were pre-treated with 50 μM NSC23766 or 20 μM SP600125 or DMSO for 24 h. Cell invasion through Matrigel was quantified as described in Materials and Methods. Shown is the mean (± SEM) of three independent experiments performed in duplicate. ** = P < 0.01 and *** = P < 0.001, two-tailed t test.