A high dose KRP203 induces cytoplasmic vacuoles associated with altered phosphoinositide segregation and endosome expansion

Abstract

In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE and VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies.

Keywords: Endosome; KRP203; Mocravimod; Phosphoinositide; RAB; Vacuolization.

Fig. 1.

A. Representative images of KRP203-induced vacuolization in cells. Cells were treated with KRP203 at 4 μM for 4 h. The unpaired two-tailed student t-test was used. B. Treatment of MEF with 10 μM S1PR1 agonist, SEW2871, for 4 h. C. Crystal violet staining of U87MG cells treated with 10 μM KRP203 for 48 h. D. Oil Red O staining of U87MG cells treated with 10 μM KRP203 for 6 h. Treatment with 0.4 mM Oleic acid/BSA was used to prepare a positive control of the staining. E. A schematic diagram of membrane trafficking. Scale bars are 50 μm.

Fig. 2.

A. Treatment of EGFP-LactC2-transfected HeLa cells with 4 μM KRP203 for 4 h. B. Treatment of EGFP-RAB5-transfected HeLa cells with 4 μM KRP203 for 4 h. C. Treatment of EGFP-FYVE2-transfected HeLa cells with 4 μM KRP203 for 6 h. The cells were immunostained with anti-EEA1 antibody. D. Treatment of EGFP-RAB11, EGFP-RAB7 or LAMP1-EGFP-transfected HeLa cells with 4 μM KRP203 for 4 h. E. Treatment of U-2 OS cells with 4 μM KRP203 for 4 h. Endogenous catalases were stained with its specific antibody. Scale bars are 20 μm.

Fig. 3.

A. Treatment of MEF with 4 μM KRP203 for 4 h. MEF were pre-treated with 100 nM Bafilomycin A1 for 1 h. Data were shown as mean ± SD. The unpaired two-tailed student t-test was used. B. Treatment of Atg5 WT or KO MEF with 4 μM KRP203 for 4 h. Data were shown as mean ± SD. The two-way ANOVA with Sidak’s multiple comparison test was used. C. Treatment of Pikfyve^flox/flox MEF with 4 μM KRP203. Pikfyve^flox/flox MEF was infected with adenovirus-Cre for 1 day and then cultured for additional 4 days to delete the Pikfyve gene (top). Treatment of MEF with 4 μM KRP203 and/or 0.8 μM YM201636 for 4 h (bottom). Time-laps images of before (0 h) and after (5 h) of the treatment are shown. D. Treatment of MEF with 4 μM KRP203 and/or 0.8 μM YM201636 for 5 h. E. Treatment of MEF with 4 μM KRP203 and/or 1 μM SAR405 for 4 h. F. Treatment of Pip4ka^−/−/Pip4kb^−/− MEF with 4 μM KRP203 for 4 h. Scale bars are 50 μm.

Fig. 4.

A. Treatment of doxycycline (DOX)-inducing PTEN U87MG cells with 4 μM KRP203 for 4 h. Cells were pretreated with 1 μg/mL DOX for 48 h. B. Treatment of PI4P-specific marker-transfected HeLa cells with 4 μM KRP203 for 4 h. Scale bars are 25 μm.