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. 2020 Feb 9;10(2):260.
doi: 10.3390/biom10020260.

UFL1 Alleviates LPS-Induced Apoptosis by Regulating the NF-κB Signaling Pathway in Bovine Ovarian Granulosa Cells

Xinling Wang  1 , Chengmin Li  1 , Yiru Wang  1 , Lian Li  1 , Zhaoyu Han  1 , Genlin Wang  1
Affiliations

UFL1 Alleviates LPS-Induced Apoptosis by Regulating the NF-κB Signaling Pathway in Bovine Ovarian Granulosa Cells

Xinling Wang et al. Biomolecules. .

Abstract

Ubiquitin-like modifier 1 ligating enzyme 1 (UFL1) is an E3 ligase of ubiquitin fold modifier 1 (UFM1), which can act together with its target protein to inhibit the apoptosis of cells. Lipopolysaccharides (LPS) can affect the ovarian health of female animals by affecting the apoptosis of ovarian granulosa cells. The physiological function of UFL1 on the apoptosis of bovine (ovarian) granulosa cells (bGCs) remains unclear; therefore, we focused on the modulating effect of UFL1 on the regulation of LPS-induced apoptosis in ovarian granulosa cells. Our study found that UFL1 was expressed in both the nucleus and cytoplasm of bGCs. The results here demonstrated that LPS caused a significant increase in the apoptosis level of bGCs in cows, and also dramatically increased the expression of UFL1. Furthermore, we found that UFL1 depletion caused a significant increase in apoptosis (increased the expression of BAX/BCL-2 and the activity of caspase-3). Conversely, the overexpression of UFL1 relieved the LPS-induced apoptosis. In order to assess whether the inhibition of bGCs apoptosis involved in the nuclear factor-κB (NF-κB) signaling pathway resulted from UFL1, we detected the expression of NF-κB p-p65. LPS treatment resulted in a significant upregulation in the protein concentration of NF-κB p-p65, and knockdown of UFL1 further increased the phosphorylation of NF-κB p65, while UFL1 overexpression significantly inhibited the expression of NF-κB p-p65. Collectively, UFL1 could suppress LPS-induced apoptosis in cow ovarian granulosa cells, likely via the NF-κB pathway. These results identify a novel role of UFL1 in the modulation of bGC apoptosis, which may be a potential signaling target to improve the reproductive health of dairy cows.

Keywords: UFL1; apoptosis; bovine granulosa cells (bGCs); lipopolysaccharide (LPS).

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Conflict of interest statement

The authors declare that they have no conflict of interests.

Figures

Figure 1
Figure 1
Ubiquitin-like modifier 1 ligating enzyme 1 (UFL1) is expressed in ovarian tissue and the cytoplasm and nucleus of granulosa cells. (a) More than 95% of the cells in the isolated cultured cells are ovarian granulosa cells. Identification of ovarian granulosa cells by immunofluorescence. (b) Immunofluorescent staining of bovine (ovarian) granulosa cells (bGCs) with anti-UFL1 antibody. (c) Bovine ovaries were analyzed by immunohistochemistry.
Figure 2
Figure 2
Lipopolysaccharide (LPS) treatment leads to increased apoptosis of ovarian granulosa cells. (a) Cell Counting Kit-8 (CCK-8) used to determine the effects of LPS on the cytotoxicity to GCs cells. (b) Effect of LPS (1 μg/mL) on the protein expression of BAX, BCL-2, and CAS3 in bGCs. Data are presented as the means ± the standard errors of the mean (SEM). ∗ p < 0.05; ∗∗ p < 0.01.
Figure 3
Figure 3
UFL1 is highly expressed in LPS-induced bGCs, and under LPS induction conditions, Ufl1 small-interfering RNA (siRNA) significantly reduced UFL1 mRNA and protein expression; overexpression of UFL1 significantly increased UFL1 expression. (a) Effect of LPS and UFL1 siRNA/UFL1 overexpression plasmid on the protein expression of UFL1 in bGCs. (b) qPCR analysis of Ufl1 in UFL1 siRNA-transfected bGCs stimulated with LPS. (c) qPCR analysis of Ufl1 in bGCs transfected with Ufl1 overexpression plasmid stimulated with LPS. Data are presented as the means ± the standard errors of the mean (SEM) of three independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.
Figure 4
Figure 4
UFL1 protects the bGCs against LPS-induced apoptosis. (a) Effect of Ufl1 siRNA on apoptosis in LPS-challenged bGCs. (b) Effect of Ufl1 overexpression on apoptosis in LPS-challenged bGCs. Data are presented as the means ± the standard errors of the mean (SEM) of three independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.
Figure 5
Figure 5
(a) Effect of Ufl1 siRNA on activity of Bax, Cas3, and Bcl-2 in LPS-challenged bGCs. (b) Effect of Ufl1 overexpression on activity of Bax, Cas3, and Bcl-2 in LPS-challenged bGCs. Data are presented as the means ± the standard errors of the mean (SEM) of three independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. (c) Representative Western blots and quantitative evaluation of BAX, CAS3, and BCL-2 in Ufl1 siRNA-transfected bGCs and Ufl1 overexpression plasmid stimulated with LPS.
Figure 6
Figure 6
UFL1 inhibits phosphorylation of NF-κB p-p65. Effect of Ufl1 siRNA and Ufl1 overexpression plasmid on the protein expression of p-p65 in bGCs. Data are presented as the means ± the standard errors of the mean (SEM) of three independent experiments. ∗ p < 0.05; ∗∗ p < 0.01.
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