Mitochondrial ROS in Slc4a11 KO Corneal Endothelial Cells Lead to ER Stress

Abstract

Recent studies from Slc4a11 ^-/- mice have identified glutamine-induced mitochondrial dysfunction as a significant contributor toward oxidative stress, impaired lysosomal function, aberrant autophagy, and cell death in this Congenital Hereditary Endothelial Dystrophy (CHED) model. Because lysosomes are derived from endoplasmic reticulum (ER)-Golgi, we asked whether ER function is affected by mitochondrial ROS in Slc4a11 KO corneal endothelial cells. In mouse Slc4a11 ^-/- corneal endothelial tissue, we observed the presence of dilated ER and elevated expression of ER stress markers BIP and CHOP. Slc4a11 KO mouse corneal endothelial cells incubated with glutamine showed increased aggresome formation, BIP and GADD153, as well as reduced ER Ca²⁺ release as compared to WT. Induction of mitoROS by ETC inhibition also led to ER stress in WT cells. Treatment with the mitochondrial ROS quencher MitoQ, restored ER Ca²⁺ release and relieved ER stress markers in Slc4a11 KO cells in vitro. Systemic MitoQ also reduced BIP expression in Slc4a11 KO endothelium. We conclude that mitochondrial ROS can induce ER stress in corneal endothelial cells.

Keywords: ERAD (ER associated protein degradation); MitoQ; ROS—reactive oxygen species; SLC4A11 ammonia transporter; corneal endothelial cells; er stress.

FIGURE 1

The ER Stress Response in WT and KO Mouse Corneal Endothelium. (A) ER stress through the unfolded protein response pathway is highly regulated through three main signaling pathways—ATF6, IRE1, and PERK. In a normal cell, the protein BIP (blue circles) is associated with the above mentioned molecules, thereby preventing the activation of ER stress associated signal transductions. With the accumulation of unfolded proteins the chaperone BIP associates with them (1) and less with the signaling molecules thereby activating the signaling pathways (2) and the ER stress associated transcriptional machinery (3). Increased GADD153 and BIP levels are considered to be two of the major outcomes of ER stress pathways (4). (B) Wes immunoassay analysis to determine the expression of ER stress markers, BIP and GADD153 in Slc4a11 WT and KO corneal endothelial tissue. (C) Quantification of Wes immunoblots from Panel (B) n = 3, **p < 0.001 (D) Electron micrograph of Slc4a11 ^+/+ and Slc4a11 ^−/− corneal endothelial tissue. Red arrows point to the ER. Scale—1 μM.

FIGURE 2

Aggresome Formation in Immortalized WT and Slc4a11 KO MCEC. (A) Flow cytometry analysis of Slc4a11 WT and KO MCEC to determine the presence of aggresomes (marker for unfolded proteins in the ER). (B) Quantification of the Geometric mean of Aggresome Intensity. Students t-test. n = 3, ****p < 0.0001.

FIGURE 3

ER Stress Response in Immortalized WT and Slc4a11 KO MCEC. (A) Wes analysis showing the expression of ER stress markers, BIP and GADD153 in Slc4a11 WT and KO corneal endothelial cells treated in media with or without glutamine. α-tubulin was used as a loading control. (B) Quantification of Wes immunoassay. Student’s t-test. n = 3, **p < 0.001, *p < 0.0 (C) Q-PCR results showing the transcript levels of spliced-XBP1 (s-XBP1), and unspliced-XBP1 (us-XBP1) levels in Slc4a11 WT and KO MCEC. Student’s t-test. n = 3, **p < 0.001.

FIGURE 4

Calcium efflux from internal stores in Immortalized WT and Slc4a11 KO MCEC. (A) Slc4a11 WT and KO cells were cultured in media without glutamine, loaded with Fura-2 and perfused with Hanks Buffer with BAPTA to establish a baseline (0–50 s), followed by perfusion with ionomycin for 100 s (B) Slc4a11 WT and KO cells were cultured in media with glutamine. The cells were perfused with Hanks Buffer with BAPTA to establish a baseline (0–50 s), followed by perfusion with ionomycin for 100 s. Student’s t-test. n = 3, **p < 0.01.

FIGURE 5

Mitochondrial ROS induces ER stress in Slc4a11 WT MCEC. Cells were treated with 0.5 μM Antimycin, 0.25 μM each of rotenone and antimycin, or 0.5 μM Rotenone for 18 h. (A) Flow cytometry analysis of mitochondrial oxidative stress using MitoSox staining. N = 3. *p < 0.05, **p < 0.01. (B) Flow cytometry analysis of apoptotic cells using Annexin V staining. N = 3, **p < 0.01, ns-not significant. (C) Flow cytometry analysis to detect the presence of aggresomes. N = 3, *p < 0.05, ns-not significant. (D,E) Wes immunoassay for BIP and quantification. N = 3, *p < 0.05, ns-not significant.

FIGURE 6

Quenching Mito ROS reverses ER stress in Slc4a11 KO. (A) Wes immunoassay for BIP and GADD153 of Slc4a11 WT and KO MCEC treated with 2 μM MitoQ. (B) Quantification of Wes immunoassay. N = 3. **p < 0.01, ***p < 0.001. Student’s t-test. (C) Flow cytometry analysis of WT and KO MCEC treated with 2 μM MitoQ to determine the presence of aggresomes. (D) Quantification of the geometric mean of aggresome intensity. N = 3. p < 0.0001. Student’s t-test. (E) Slc4a11 WT and KO MCEC were cultured in media with glutamine ± 2 μM MitoQ. The cells were loaded with Fura-2 and perfused with Hanks Buffer with BAPTA to establish a baseline (0–50 s), followed by perfusion with ionomycin for 100 s. (F) Wes immunoassay for BIP in Slc4a11 WT and KO corneal endothelial tissue of animals treated with or without MitoQ. (G) Quantification of Wes immunoassay. N = 3. **p < 0.01, ns-not significant.

FIGURE 7

Proposed mechanism for ER stress in Slc4a11 ^−/− corneal endothelium. Elevated mitochondrial ROS leads to ER stress. This results in decreased ER Ca2+ levels, elevated levels of aggresomes and increase in Unfolded Protein Pathway. Use of mitochondrial ROS quencher, MitoQ, alleviates ER stress in Slc4a11 ^−/− corneal endothelium.