EZH2 Gene Knockdown Inhibits Sheep Pituitary Cell Proliferation via Downregulating the AKT/ERK Signaling Pathway

Abstract

Pituitary gonadotropins perform essential functions in mammalian reproduction by stimulating gametogenesis and steroidogenesis in the ovaries and testicles. EZH2 is a histone methyltransferase that inhibits proliferation and aggravates apoptosis in stem cells subjected to pathological stimuli. However, the expression and molecular mechanisms of EZH2 in pituitary cells in vitro have not been extensively studied. In this study, the relative abundances of EZH2 mRNA (p < 0.01) and protein (p < 0.05) expression were larger in the pituitary cells of Hu sheep with relatively greater fecundity (GF) compared to those with lesser fecundity (LF). Loss-of-function examinations demonstrated that EZH2 gene knockdown led to an earlier induction of apoptosis in sheep pituitary cells (PCs). The relative abundance of CASP3, CASP9, and BAX was increased (p < 0.01), while BCL2's abundance was less decreased (p < 0.01) in PCs where there was EZH2 gene knockdown. Additionally, cell proliferation (p < 0.01) and viability (p < 0.01) were decreased in EZH2-knockdown sheep PCs, and the cell cycle was blocked compared to a negative control (NC). Notably, EZH2 gene knockdown led to reduced abundances of gonadotropin subunit gene transcripts (FSHβ, p < 0.05) and reduced FSH release (p < 0.01) from PCs. EZH2 gene knockdown led to reduced phosphorylation of AKT, ERK, and mTOR (p < 0.01). The results suggest that EZH2 regulates pituitary cell proliferation, apoptosis, and FSH secretion through modulation of the AKT/ERK signaling pathway, providing a foundation for further study of pituitary cell functions.

Keywords: EZH2; Hu sheep; apoptosis; gonadotropin secretion; pituitary cells; proliferation.

Figure 1

EZH2 is present in the Hu sheep pituitary cells: (A) Relative mRNA abundance of EZH2 was determined in the HPO axis of Hu sheep by qPCR, and β-actin was used as an internal control. (B) The relative abundance of EZH2, depicted at different developmental stages (90 fetus, 120 fetus, 0, 5 days, 3 months, 6 months, 8 months, and 2 years after birth) of the pituitary from Hu sheep; β-actin was used as an internal control. (C) EZH2 had greater relative abundance in the pituitary cells from Hu sheep with relatively greater fecundity (GF) in comparison to those with lesser fecundity (LF), and β-actin was used as an internal control. (D) Immunocytochemistry for EZH2 in Hu sheep pituitary cells relative to negative controls (NC). Scale bars correspond to 100 μm. (E) There was a greater relative abundance of EZH2 protein in the pituitary cells from Hu sheep with relatively greater fecundity (GF) relative to those with lesser fecundity (LF), as determined using Western blot procedures. Data are reported as the mean ± SEM (** p < 0.01). The values with different letters a and b indicate statistically significant differences (p-value < 0.05).

Figure 2

EZH2 mRNA transcripts had greater abundance in GnRH-stimulated pituitary cells: (A) EZH2’s location in Hu sheep pituitary cells when using the cytoplasmic and nuclear RNA fractionation assay. (B) Immunofluorescence with EZH2 antibody indicating that EZH2 is located in the nucleus and cytoplasm of pituitary cells. Scale bars correspond to 20 μm. The average signal intensity was determined using ImageJ 1.42q software (http://rsb.info.nih.gov/ij, accessed on 23 June 2023). The DAPI results indicate nuclear staining. Merged results are indicative of where there were staining overlays. Suppression of the relative abundance of EZH2 in pituitary cells using si-EZH2, as confirmed using qPCR (C) and Western blot (D). Increased mRNA transcription of EZH2 was determined in GnRH-stimulated pituitary cells, as measured by qPCR (E) and Western blot procedures (F). Data are reported as the mean ± SEM (* p < 0.05, ** p < 0.01).

Figure 3

EZH2 inhibition limits the proliferation and cell cycle of PCs’ functions: (A) Downregulation of EZH2 gene expression induced an arrest of the cell cycle at the S phase in PCs. Vertical axis: % of cells. (B) CCK-8 results indicating that the suppression of EZH2 reduced the proliferation potential of PCs. (C) Sheep PCs treated with siEZH2 had reduced Edu incorporation compared to the NC. Hoechst staining results indicated that EZH2 was present in the nucleus. Scale bars correspond to 100 μm. (D) Relative abundances of PCNA, CDK2, CDK4, CCND1, and CCNE2 mRNA transcripts were less in EZH2-knockdown sheep PCs compared to the NC, as determined using qPCR. (E) The relative abundances of PCNA and CCND1 were reduced as a result of silencing of the EZH2 gene. Data are reported as the mean ± SEM (* p < 0.05, ** p < 0.01).

Figure 4

Knockdown of the EZH2 gene led to greater apoptosis in PCs: (A,B) Downregulation of EZH2 gene expression led to enhanced apoptosis in sheep PCs. (C) Relative abundances of BAX, BCL2, CASP3, and CASP9 were greater in PCs where there was downregulation of EZH2 gene expression in comparison with PCs of the control group (NC). (D) Relative abundances of total PARP, cleaved PARP, BAX, and BCL2 were altered when there was downregulation of EZH2 gene expression. Data are reported as the mean ± SEM (* p < 0.05, ** p < 0.01).

Figure 5

EZH2 gene silencing led to decreased FSH secretion from PCs: (A) EZH2 gene downregulation resulted in reduced FSH secretion from sheep PCs. (B) The relative abundance of LHβ, FSHβ, and CGA mRNA transcripts was less in PCs when there was EZH2 gene knockdown in sheep PCs compared to the PCs of the NC group. (C) There was less FSHβ in PCs when there was EZH2 gene knockdown. Data are reported as the mean ± SEM (* p < 0.05, ** p < 0.01).

Figure 6

EZH2 may have functions in the AKT/ERK-mTOR pathways: (A) The relative abundance of PI3K, AKT1, AKT2, AKT3, and mTOR in PCs when there was EZH2 gene silencing. (B) Relative abundances of ERK, AKT, and mTOR proteins and their phosphorylation in PCs of sheep where there was EZH2 gene knockdown, compared with the NC group. Data are reported as the mean ± SEM (* p < 0.05, ** p < 0.01).