Protein Kinase A and 5' AMP-Activated Protein Kinase Signaling Pathways Exert Opposite Effects on Induction of Autophagy in Luteal Cells.

Emilia Przygrodzka,Michele R Plewes,John S Davis,Corrine F Monaco,Jennifer R Wood,Andrea S Cupp,Guojuan Li

doi:10.3389/fcell.2021.723563

Abstract

In the absence of pregnancy the ovarian corpus luteum undergoes regression, a process characterized by decreased production of progesterone and structural luteolysis involving apoptosis. Autophagy has been observed in the corpus luteum during luteal regression. Autophagy is a self-degradative process important for balancing sources of cellular energy at critical times in development and in response to nutrient stress, but it can also lead to apoptosis. Mechanistic target of rapamycin (MTOR) and 5′ AMP-activated protein kinase (AMPK), key players in autophagy, are known to inhibit or activate autophagy, respectively. Here, we analyzed the signaling pathways regulating the initiation of autophagy in bovine luteal cells. In vivo studies showed increased activating phosphorylation of AMPKα (Thr172) and elevated content of LC3B, a known marker of autophagy, in luteal tissue during PGF2α-induced luteolysis. In vitro, AMPK activators 1) stimulated phosphorylation of regulatory associated protein of MTOR (RPTOR) leading to decreased activity of MTOR, 2) increased phosphorylation of Unc-51-Like Kinase 1 (ULK1) and Beclin 1 (BECN1), at sites specific for AMPK and required for autophagy initiation, 3) increased levels of LC3B, and 4) enhanced colocalization of autophagosomes with lysosomes indicating elevated autophagy. In contrast, LH/PKA signaling in luteal cells 1) reduced activation of AMPKα and phosphorylation of RPTOR, 2) elevated MTOR activity, 3) stimulated phosphorylation of ULK1 at site required for ULK1 inactivation, and 4) inhibited autophagosome formation as reflected by reduced content of LC3B-II. Pretreatment with AICAR, a pharmacological activator of AMPK, inhibited LH-mediated effects on RPTOR, ULK1 and BECN1. Our results indicate that luteotrophic signaling via LH/PKA/MTOR inhibits, while luteolytic signaling via PGF2α/Ca2+/AMPK activates key signaling pathways involved in luteal cell autophagy.

Highlights

The corpus luteum is a transient endocrine gland crucial for the establishment and maintenance of pregnancy
To confirm that prostaglandin F2α (PGF2α) triggers AMPK, bovine large luteal cells were incubated in the presence of PGF2α for 2–30 min
Knowing that AMPK can be activated by calcium/ calmodulin dependent protein kinase kinase 2 (CAMKK2), a kinase evoked by PGF2α in bovine luteal tissue (Davis et al, 1987; Bowdridge et al, 2015; Herzig and Shaw 2018), we pretreated luteal cells with inhibitor of CAMKK2, STO-609, and treated them with PGF2α

Summary

Introduction

The corpus luteum is a transient endocrine gland crucial for the establishment and maintenance of pregnancy. Luteinizing hormone (LH), a gonadotropin produced by the pituitary gland, regulates the formation, development, and maintenance of the corpus luteum (Davis and Rueda 2002; Berisha and Schams 2005). A main function of the corpus luteum is to produce progesterone, a steroid hormone that regulates embryo development, reduces uterine contractions, suppresses maternal immune response, prepares endometrium for implantation, prevents preeclampsia, and regulates the estrous/ menstrual cycle (Davis and Rueda 2002; Park et al, 2020; Pereira et al, 2021). In the absence of an embryo, the corpus luteum undergoes regression, a process characterized by decreased production of progesterone and structural luteolysis (Juengel et al, 1993; Rueda et al, 1997; McCracken et al, 1999), resulting in the formation of a fibrotic scar called the corpus albicans. The presence of live embryos in the uterus extends the lifespan of the corpus luteum due to the luteotrophic and anti-luteolytic actions of conceptus signals (Davis and Rueda 2002)

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