The Activation of Reticulophagy by ER Stress through the ATF4-MAP1LC3A-CCPG1 Pathway in Ovarian Granulosa Cells Is Linked to Apoptosis and Necroptosis

Abstract

Female infertility is caused by premature ovarian failure (POF), which is triggered by the endoplasmic reticulum (ER) stress-mediated apoptosis of granulosa cells. The ER unfolded protein response (UPR^er) is initiated to promote cell survival by alleviating excessive ER stress, but cellular apoptosis is induced by persistent or strong ER stress. Recent studies have reported that reticulophagy is initiated by ER stress. Whether reticulophagy is activated in the ER stress-mediated apoptosis of granulosa cells and which pathway is initiated to activate reticulophagy during the apoptosis of granulosa cells are unknown. Therefore, the role of reticulophagy in granulosa cell death and the relationship between ER stress and reticulophagy were investigated in this work. Our results suggest that the ER stress inducer tunicamycin causes POF in mice, which is attributed to the apoptosis of granulosa cells and is accompanied by the activation of UPR^er and reticulophagy. Furthermore, granulosa cells were treated with tunicamycin, and granulosa cell apoptosis was triggered and increased the expression of UPR^er and reticulophagy molecules. The expression of ATF4 was then downregulated by RNAi, which decreased the levels of autophagy and the reticulophagy receptor CCGP1. Furthermore, ATF4 targets MAP1LC3A, as revealed by the ChIP sequencing results, and co-IP results demonstrated that MAP1LC3A interacts with CCPG1. Therefore, reticulophagy was activated by ER stress through the ATF4-MAP1LC3A-CCPG1 pathway to mitigate ER stress. Additionally, the role of reticulophagy in granulosa cells was investigated by the knockdown of CCPG1 with RNAi. Interestingly, only a small number of granulosa cells died by apoptosis, whereas the death of most granulosa cells occurred by necroptosis triggered by STAT1 and STAT3 to impair ER proteostasis and the ER protein quality control system UPR^er. Taken together, the results indicate that the necroptosis of granulosa cells is triggered by up- and downregulating the reticulophagy receptor CCPG1 through STAT1/STAT3-(p)RIPK1-(p)RIPK3-(p)MLKL and that reticulophagy is activated by ER stress through the ATF4-MAP1LC3A-CCPG1 pathway.

Keywords: CCPG1; ER stress; apoptosis; granulosa cells; necroptosis; reticulophagy; unfolded protein response.

Figure 1

Reticulophagy activated by the ER stress inducer tunicamycin causes POF. (A): Histology of ER stress inducer tunicamycin (Tm)-treated mouse ovaries; (B): Ultrastructure of oocytes and granulosa cells (GCs) in primary, secondary, and tertiary follicles by transmission electron microscopy detection; (C): Western blot analysis of ovaries from control and 5 μg/g tunicamycin-treated mice. β-actin was used as a reference protein, *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 2

Reticulophagy activated by the ER stress inducer tunicamycin causes granulosa cell apoptosis. (A): Detection of the UPR^er and reticulophagy by Western blotting and analysis of total protein in the control and 5 μM tunicamycin-treated granulosa cells; (B): Detection of the UPR^er by Western blotting and analysis of nuclear protein in the control and 5 μM tunicamycin-treated granulosa cells; (C): Detection of cellular apoptosis by flow cytometry in the control and 5 μM tunicamycin-treated granulosa cells; (D): Detection of the UPR^er molecule ATF4 and reticulophagy by Western blotting and analysis of total protein in the control, 5 μM tunicamycin-, 5 Mm+4-PBA-, and 5 μM tunicamycin+5 mM 4-PBA-treated granulosa cells; (E): Detection of the UPR^er molecule ATF4 and reticulophagy by Western blotting and analysis of total protein in the control, 5 μM tunicamycin-, 10 mM 3-MA-, and 5 μM tunicamycin+10 mM 3-MA-treated cells. β-actin was used as a reference protein, *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 3

Immunofluorescence of markers of the UPR^er and autophagy in control and 5 μM tunicamycin-treated cells. (A): Colocalization of ATF4 (green) protein and ER tracker (red); (B): Colocalization of CHOP (green) protein and ER tracker (red); (C): Colocalization of XBP1s (green) protein and ER tracker (red); (D): Colocalization of ATF6 (green) protein and ER tracker (red); (E): Colocalization of ATF4 (red) protein and GFP-LC3 (green); (F): Colocalization of CCPG1 (red) protein and GFP-LC3 (green); (G): Autophagic flux detection by mRFP-GFP-LC3B staining. At least 50 different cells from different fields were analyzed, and the dots were blindly counted by three different persons. The nuclei were stained with DAPI (blue). * p < 0.05.

Figure 4

ATF4 targets MAP1LC3/CCPG1. (A): Heatmap of control and 5 μM tunicamycin-treated cells generated from the ChIP sequencing results; (B): Enriched KEGG pathway analysis of the ChIP sequencing results; (C): Binding site of ATF4 in MAP1LC3A; (D): Detection of reticulophagy by Western blotting and results of ATF4 inhibition in 5 μM tunicamycin-treated cells; (E): MAP1LC3A interacts with CCPG1, as revealed by co-IP; (F): Colocalization of CCPG1 (red) protein and GFP-LC3 (green); (G): Autophagic flux detection by mRFP-GFP-LC3B staining. The nuclei stained with DAPI (blue). At least 50 different cells in different fields were analyzed, and the dots were blindly counted by three different individuals. * p < 0.05.

Figure 5

Impaired ER proteostasis in CCPG1-knockdown cells. (A): Detection of the UPR^er, reticulophagy and apoptotic marker molecules by Western blotting in CCPG1-knockdown granulosa cells; (B): Colocalization of ATF4 (green), CHOP (green), XBP1s (green), ATF6 (green), and ER tracker (red) in CCPG1-knockdown granulosa cells; (C): Detection of cellular apoptosis by flow cytometry in the control and CCPG1-knockdown cells; (D): Protein aggregate detection results. At least 50 different cells in different fields were analyzed, and the dots were blindly counted by three different individuals. β-Actin was used as a reference protein, *** p < 0.001, ** p < 0.01, * p < 0.05. The nuclei are stained with DAPI (blue).

Figure 6

Activation of necroptosis by downregulation of CCPG1 by STAT1 and STAT3. (A): Results from the detection of necroptosis by Western blotting in CCPG1-knockdown granulosa cells; (B): Detection of necroptosis by Western blotting in granulosa cells treated with the necroptosis inhibitor necrostatin-1; (C): Detection of necroptosis and STAT1 by Western blotting in granulosa cells treated with the STAT1 inhibitor fludarabine; (D): Detection of necroptosis and STAT1 by Western blotting in granulosa cells treated with the STAT3 inhibitor niclosamide; (E): Colocalization of STAT1 (Green) and ER tracker (Red) in CCPG1-knockdown cells; (F): Colocalization of STAT3 (Green) and ER tracker (Red) in CCPG1-knockdown cells; (G): CCPG1 directly interacts with STAT1, as revealed by co-IP detection; (H): STAT1 and STAT3 directly interact with ripk1, as revealed by co-IP detection. β-Actin was used as a reference protein, * p < 0.05.

Figure 7

Pathway through which CCPG1 in necroptosis is linked to ER stress-mediated apoptosis. The green line indicates ER stress, the UPR^er, and autophagy; the red line indicates necroptosis; the yellow line indicates apoptosis.