Lipotoxicity-Induced PRMT1 Exacerbates Mesangial Cell Apoptosis via Endoplasmic Reticulum Stress

Abstract

Lipotoxicity-induced mesangial cell apoptosis is implicated in the exacerbation of diabetic nephropathy (DN). Protein arginine methyltransferases (PRMTs) have been known to regulate a variety of biological functions. Recently, it was reported that PRMT1 expression is increased in proximal tubule cells under diabetic conditions. However, their roles in mesangial cells remain unexplored. Thus, we examined the pathophysiological roles of PRMTs in mesangial cell apoptosis. Treatment with palmitate, which mimics cellular lipotoxicity, induced mesangial cell apoptosis via protein kinase RNA-like endoplasmic reticulum kinase (PERK) and ATF6-mediated endoplasmic reticulum (ER) stress signaling. Palmitate treatment increased PRMT1 expression and activity in mesangial cells as well. Moreover, palmitate-induced ER stress activation and mesangial cell apoptosis was diminished by PRMT1 knockdown. In the mice study, high fat diet-induced glomerular apoptosis was attenuated in PRMT1 haploinsufficient mice. Together, these results provide evidence that lipotoxicity-induced PRMT1 expression promotes ER stress-mediated mesangial cell apoptosis. Strategies to regulate PRMT1 expression or activity could be used to prevent the exacerbation of DN.

Keywords: ER stress; PRMT1; apoptosis; lipotoxicity; mesangial cell.

Figure 1

Lipotoxicity induces mesangial cell apoptosis via protein kinase RNA-like endoplasmic reticulum kinase (PERK) and ATF6 signaling pathways. (A,B) Mesangial cells were treated with 30 μM palmitate for various time intervals. Cell extracts were subjected to western blot analysis with indicated antibodies. The representative immunoblots were from at least three independent experiments; (C) mesangial cells were treated with 50 nM thapsigargin for 6 h and 12 h. Cell extracts were subjected to western blot analysis with caspase-3 antibody. The representative immunoblots were from at least three independent experiments; (D–F) mesangial cells were pretreated with 1 μM GSK2606414 or 100 μM AEBSF. After 30 min, cells were treated with 30 μM palmitate for 24 h; (D) cell extracts were subjected to western blot analysis with indicated antibodies. The representative immunoblots were from at least three independent experiments; (E) caspase 3/7 activity was measured. Data represent the mean ± SEM of three independent experiments, each performed in triplicate. * p < 0.05 vs. control, ** p < 0.05 vs. palmitate; (F) annexin V and propidium iodide stainings were analyzed. Red frame indicates the sum of early and late apoptotic cells. Representative images were from at least three independent experiments.

Figure 2

Protein arginine methyltransferase 1 (PRMT1) expression is increased in palmitate-treated mesangial cells. (A) Mesangial cells were treated with 30 μM palmitate for various time intervals. Type I PRMT expressions were assayed by western blot analysis. The representative immunoblots were from at least three independent experiments. Data represent the means ± SEM of three independent experiments. * p < 0.05 vs. 0 h (ROD: relative optical density); (B) mesangial cells were treated with 30 μM palmitate for 24 h. The cells were labeled with anti-PRMT1 and a fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and observed under a confocal microscope. The representative images were from at least three independent experiments (scale bar: 5 μm); (C) mesangial cells were treated with 30 μM palmitate for 12 h and 24 h. Cell extracts were subjected to western blot analysis with ASYM24 antibody. Representative immunoblots were from at least three independent experiments.

Figure 3

PRMT1 knockdown attenuates palmitate-induced ER stress signaling and mesangial cell apoptosis. (A) Mesangial cells were transfected with scramble or PRMT1 siRNA according to the reverse transfection method. After 36 h, cell extracts were subjected to western blot analysis with indicated antibodies. The representative immunoblots were from at least three independent experiments; (B–E) mesangial cells were transfected with scramble or PRMT1 siRNA according to the reverse transfection method. After 24 h, cells were treated with 30 μM palmitate for 24 h; (B,C) cell extracts were subjected to western blot analysis with indicated antibodies. Representative immunoblots were from at least four independent experiments; (D) caspase 3/7 activity was measured. Data represent the mean ± SEM of three independent experiments, each performed in triplicate. * p < 0.05 vs. scramble, ** p < 0.05 vs. scramble + palmitate; (E) annexin V and propidium iodide stainings were analyzed. Red frame indicates the sum of early apoptotic cells and late apoptotic cells. The representative images were from at least three independent experiments.

Figure 4

High fat diet (HFD)-induced glomerular apoptosis is attenuated in PRMT1 haploinsufficient mice. (A) Kidney sections were subjected to terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay as described in “Materials and Methods”, (scale bar: 20 μM); (B) summarized scheme; lipotoxicity-induced PRMT1 exacerbates mesangial cell apoptosis via ER stress.