Elsevier

Cellular Signalling

Volume 73, September 2020, 109702
Cellular Signalling

TNFα-Erk1/2 signaling pathway-regulated SerpinE1 and SerpinB2 are involved in lipopolysaccharide-induced porcine granulosa cell proliferation

https://doi.org/10.1016/j.cellsig.2020.109702Get rights and content

Highlights

  • LPS increases porcine granulosa cell proliferation.

  • LPS-stimulated TNFα is a crucial factor in porcine granulosa cell proliferation.

  • LPS TNFα stimulated porcine granulosa cell proliferation via SerpinE1 and SerpinB2 upregulation.

  • Erk1/2 signaling pathway plays crucial roles in LPS- and TNFα-induced SerpinE1 and SerpinB2 upregulation and GC proliferation.

  • LPS- and TNFα-induced MMP polarization and ATP production play synergistic effects on GC proliferation.

Abstract

Lipopolysaccharide (LPS) is an inhibitory factor that causes hormonal imbalance and subsequently affects ovarian function and fertility in mammals. Previous studies have shown that the exposure of granulosa cells (GC) to LPS leads to steroidogenesis dysfunction. However, the effects of LPS on the viability of GC remain largely unclear. In the present study, we aimed to address this question and unveil the underlying molecular mechanisms using cultured porcine GC. Results showed that GC proliferation and tumor necrosis factor α (TNFα) secretion were significantly increased after exposure to LPS, and these effects were completely reversed by blocking the TNFα sheddase, ADAM17. Moreover, GC proliferation induced by LPS was mimicked by treatment with recombinant TNFα. In addition, SerpinE1 and SerpinB2 expression levels increased in GC after treatment with LPS or recombinant TNFα, whereas blocking the Erk1/2 pathway completely abolished these effects and also inhibited GC proliferation. Further, consistent with the effects of blocking the Erk1/2 pathway, cell proliferation was completely inhibited by knocking down SerpinE1 or SerpinB2 in the presence of LPS or recombinant TNFα. Mitochondrial membrane potential (MMP) polarization in GC was increased by LPS or recombinant TNFα treatment, and these changes were completely negated by Erk1/2 inhibition, but not by SerpinE1 or SerpinB2 knockdown. Taken together, these results suggested that the TNFα-mediated upregulation of SerpinE1 and SerpinB2, through activation of the Erk1/2 pathway plays a crucial role in LPS-stimulated GC proliferation, and the increase in GC MMP may synergistically influence this process.

Introduction

Granulosa cells (GC) are the predominant somatic cells in mammalian ovarian follicles. They constitute the major functional component of mammalian ovarian follicles and produce sex steroid hormones to provide nourishment to the oocyte [[1], [2], [3], [4]]. During the development of ovarian follicles, there is an increase in the number of GC (proportional to the developmental stage; from a monolayer in primordial follicles to multilayer in antral follicles) in response to gonadotropins and intrafollicular growth factors [[5], [6], [7]]. Otherwise, GC apoptosis triggers follicular atresia in mammalian ovaries [8]. Indeed, numerous in vitro and in vivo studies have shown the close correlation between GC apoptosis and fertility problems in mammals [9,10]. However, abnormal GC proliferation may also impair mammalian fertility. For example, previously reported clinical studies have shown that GC proliferation increases in the ovaries of women suffering from anovulatory polycystic ovary syndrome (PCOS) [11,12], and this increase in GC proliferation has also been observed in primate PCOS models [13]. Collectively, these reports suggest that tightly controlled GC proliferation is essential for normal follicular development during mammalian reproduction.

Bacterial infection, which commonly occurs in animals and humans, is an important factor that impairs ovarian function and fertility. Importantly, gram-negative bacteria are major causative agents of infection towing to their endotoxins, such as lipopolysaccharide (LPS), the main component of the bacterial outer membrane [14]. Indeed, LPS can be detected in the serum and even in the follicular fluid of animals and human patients with bacterial infections [15,16]. Previous studies have shown that the LPS concentration in the follicular fluid of healthy animals is approximately 0.06 ng·mL−1, while its concentration can be significantly increased to 176.1 ± 112 ng·mL−1 and in some cases, up to 875.2 ng·mL−1, in animals with bacterial infections [17]. Moreover, it is well established that pathogen-associated molecular patterns recognition receptors, such as toll-like receptors, especially TLR4, which is the specific receptor of LPS, are expressed on GC surface, and respond directly to LPS stimulation [17]. Therefore, LPS causes hormonal imbalance and subsequent reproduction failure in animals by perturbing the function of GC [18]. In support of this, previous research in our lab and other labs has demonstrated that LPS suppresses estrogen production, decreases the expression level of gonadotropin receptors and the aromatase gene CYP19A1, in GC [17,[19], [20], [21]]. Thus, it is commonly accepted that LPS impairs GC function. However, the effects of LPS on GC viability remain largely unclear.

Serpin family E member 1 (SerpinE1, also known as plasminogen activator inhibitor-1, PAI-1) and serpin family B member 2 (SerpinB2, also known as plasminogen activator inhibitor-2, PAI-2) are specific inhibitors of urokinase-type plasminogen activator (uPA), which is an efficient enzyme involved in tissue remodeling [22]. These two inhibitors are synthesized by several cell types and are known to promote cell survival [[23], [24], [25]]. Numerous studies on cancer have suggested that the overexpression of SerpinE1 is related to cell proliferation, tumor invasion and aggressiveness [26]. Additionally, SerpinB2 overexpression is known to inhibit apoptosis, promote cell survival [27], and confers protection against cytolysis in response to viral or bacterial infections [28,29]. High level of SerpinE1 expression have been detected in ovarian tissues of PCOS patients [30] and transgenic mouse models that constitutively and stably express SerpinE1 exhibit pathophysiological characteristics of human PCOS [31]. Taken together, these observations suggest that SerpinE1 and SerpinB2 may be involved in GC proliferation. Therefore, in the present study, we aimed to investigate the effects of LPS on SerpinE1 and SerpinB2 expression, GC proliferation, and to determine the underlying molecular mechanisms.

Section snippets

Granulosa cell isolation and culture

Porcine ovaries were obtained from slaughterhouse and transported to the laboratory in sterile physiological saline at 37 °C within 2 h. The ovaries were washed at least three times with preheated sterile physiological saline (37 °C). Thereafter, follicular fluid and GCs were aspirated from medium-sized healthy follicles by using syringe. The cells were then purified by density centrifugation as described elsewhere [32]. Subsequently centrifuged at 2000 rpm for 3 min to precipitate the cells

LPS increased porcine GC proliferation

To investigate the effect of LPS on GC proliferation, first, a cell counting kit-8 (CCK-8) assay was used to quantitate cell viability after treatment with serially increasing concentrations of LPS (500 ng·mL−1, 1000 ng mL−1,and 2000 ng mL−1) for 24 h. As shown in Fig. 1A, cell viability was significantly increased by LPS in a dose-dependent manner. To confirm these observations, we adopted flow cytometric approaches. As shown in Fig. 1B and C, the number of viable cells was found to increase

Discussion

Numerous studies have established that LPS inhibits GC function by reducing steroidogenesis and gonadotropin receptor expression levels [21,40,41]. Additionally, LPS-induced cell proliferation has been confirmed in several other cell types [39,[42], [43], [44], [45], [46]]. However, until now, few studies have been reported on the effects of LPS on GC proliferation. In this study, we reported for the first time that porcine GC proliferation significantly increased in response to LPS, and that

Founding statement

This study was supported by the National Natural Science Foundation of China (grant no. 31402080 and 31601943), and Natural Science Foundation of Jiangsu Province (grant no. BK20151365).

Credit author statement

Conceptualization: Hui Li, Data curation:Xiaolu Qu, Shuangshuang Guo, Leyan Yan, Huanxi Zhu, Formal analysis:Xiaolu Qu, Hui Li, Funding acquisition: Hui Li, Leyan Yan, Investigation: Hui Li, Zhendan Shi, Methodology:Hui Li, Zhendan Shi , Project administration: Hui Li,Supervision:Zhendan Shi, Validation:Hui Li, Original draft:Hui Li, Review & editing: Zhendan Shi.

Declaration of Competing Interest

The authors declare no conflict of interest.

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