Rho GTPases are involved in S1P‐enhanced glomerular endothelial cells activation with anti‐myeloperoxidase antibody positive IgG

Abstract Sphingosine‐1‐phosphate (S1P) is a crucial regulator in vascular inflammation. Our recent study found that under pathophysiological concentration in active anti‐neutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV), S1P participated in MPO‐ANCA‐positive IgG‐induced glomerular endothelial cell (GEnC) activation via a S1P receptor (S1PR)‐dependent way. However, the downstream signalling pathways are not fully clear yet. In this study, we demonstrated that Rho guanosine triphosphatases (GTPases) signalling pathways, RhoA and Rac1 in particular, were implicated in MPO‐ANCA‐positive IgG‐mediated GEnCs activation enhanced by pathophysiological concentration of S1P in AAV. These results provide mechanistic insights into vascular barrier dysfunction in AAV, which may facilitate the development of effective therapies.

ment of AAV, 6 and S1P participated in GEnC activation induced by MPO-ANCA-positive immunoglobulin (Ig)G via S1PR2-5. 7 However, the downstream signalling pathways of S1P in this process are still not fully clear.

Rho guanosine triphosphatases (GTPases) mediate diverse biolog-
ical responses including morphogenesis, chemotaxis and cell cycle progression. 8 It was reported that Rho GTPases, especially Rac1 and RhoA, could regulate endothelial barrier function in response to S1P and its receptors. S1P of physiological level causes the activation of S1PR1, resulting in protection of the endothelial barrier function by inducing the activation of the Rac1 signalling pathway, whereas excessive S1P will bind to S1PR2/S1PR3, leading to the activation of RhoA as well as the disruption of endothelial barrier function. [9][10][11] In view of the potential role of Rho GTPases in regulating endothelial barrier function, we hypothesized that Rho GTPases, RhoA and Rac1 in particular, might contribute to S1P-enhanced GEnC activation in the presence of MPO-ANCA-positive IgG.

| Reagents
See the "Supporting Information Data S1."

| Cell culture
Primary human glomerular endothelial cells (GEnC; ScienCell, San Diego, CA, USA) were cultured according to the manufacturer's instructions.

| IgG preparation
MPO-ANCA-positive IgGs and normal IgGs were prepared as previously described 7 (detailed in the "Supporting Information Data S1").

| Measurement of Rho GTPase activation
Rac1 and RhoA activation assays were performed following the manufacturer's instructions (Cytoskeleton, Denver, CO, USA).

| Statistical analysis
The normality of our data was evaluated by skewness and kurtosis (both the absolute values were less than 3). Differences were considered statistically significant if P < .05 (detailed in the "Supporting Information Data S1").

GTPases in GEnCs stimulated by S1P plus MPO-ANCA-positive IgG
Pre-incubation of GEnCs with the S1PR1 selective agonist SEW could significantly increase the activity of Rac1 in GEnCs treated by S1P plus MPO-ANCA-positive IgG, with the increase rate of 16.2 AE 6.5%, while the inhibition of S1PR1 with the antagonist W146 attenuated the Rac1 activity in GEnCs treated by S1P combined with MPO-ANCA-positive IgG by 26.9 AE 7.3%. On the contrary, compared with that without the antagonist, the S1PR2-4 selective antagonists JTE, TY and CYM significantly down-regulated the relative activity of RhoA in GEnCs treated by S1P plus MPO-ANCA-positive IgG (expressed as percentages of the control, 120.5 AE 2.9% vs 147.9 AE 5.2%, P < .001 by ANOVA; 113.9 AE 9.4% vs 147.9 AE 5.2%, P < .001 by ANOVA; 124.7 AE 4.4% vs 147.9 AE 5.2%, P < .001 by ANOVA, respectively), while the S1PR5 agonist A97 up-regulated the RhoA activity (181.9 AE 6.6% vs 147.9 AE 5.2%, P < .001 by ANOVA, with the increase rate of 23.0 AE 4.5%) ( Figure 1C,D). These results indicated that S1P,

| DISCUSSION
In our present study, we demonstrated that under pathophysiological concentration in active AAV patients, S1P could activate both RhoA and Rac1 signalling pathways in MPO-ANCA-positive IgG-treated GEnCs. According to Singleton et al, RhoA and Rac1 play opposing roles in regulating endothelial barrier function in response to differential activation of S1PRs. 11 RhoA activated by S1PR2/3 disrupts endothelial barrier function by enhancing the formation of contractile stress fibres which connect to junctions and generate pulling forces within neighbouring cells, therefore inducing destabilization of cell contact and internalization of molecules in tight junctions and adherent junctions. 13 Loss of endothelial cell-cell contact and increased permeability also facilitates leukocyte transendothelial migration and damage to endothelium, which is of vital importance in AAV. 14 Contrary to RhoA, Rac1 activated by S1PR1 enhances endothelial barrier function by inducing reorganization of the actin cytoskeleton as well as affecting the formation of lamellipodia and membrane ruffles. 15 In the present study, we found that RhoA activated by S1PR2-5 dominated the S1P- In conclusion, our current study demonstrated that Rho GTPases signalling pathways were involved in S1P-enhanced GEnCs activation with MPO-ANCA-positive IgG. RhoA activated by S1PR2-5 dominated the S1P-induced GEnC activation in the presence of MPO-ANCA-positive IgG, while Rac1 activated by S1PR1 exerted opposite effect during this process. These findings provide us with more clues to determine the role of and Rho GTPases and S1P in the development of AAV.