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

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.

SUBMITTER: Przygrodzka E

PROVIDER: S-EPMC8607825 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AIMS:The abundance of beta 3-adrenergic receptors (?3-ARs) is upregulated in diseased human myocardium. We previously showed that cardiac-specific expression of ?3-AR inhibits the hypertrophic response to neurohormonal stimulation. Here, we further analysed signalling pathways involved in the anti-hypertrophic effect of ?3-AR. METHODS AND RESULTS:In vitro hypertrophic responses to phenylephrine (PE) were analysed in neonatal rat ventricular myocytes (NRVM) infected with a recombinant adenovirus expressing the human ?3-AR (AdVh?3). We confirmed results in mice with cardiomyocyte-specific moderate expression of human ?3-AR (?3-TG) and wild-type (WT) littermates submitted to thoracic transverse aortic constriction (TAC) for 9 weeks. We observed a colocalization of ?3-AR with the AMP-activated protein kinase (AMPK) both in neonatal rat and in adult mouse cardiomyocytes. Treatment of NRVM with PE induced hypertrophy and a decrease in phosphorylation of Thr172-AMPK (/2, P = 0.0487) and phosphorylation of Ser79-acetyl-CoA carboxylase (ACC) (/2.6, P = 0.0317), inducing an increase in phosphorylated Ser235/236 S6 protein (×2.5, P = 0.0367) known to be involved in protein synthesis. These effects were reproduced by TAC in WT mice but restored to basal levels in ?3-AR expressing cells/mice. siRNA targeting of AMPK partly abrogated the anti-hypertrophic effect of ?3-AR in response to PE in NRVM. Concomitant with hypertrophy, autophagy was decreased by PE, as measured by microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I ratio (/2.6, P = 0.0010) and p62 abundance (×3, P = 0.0016) in NRVM or by TAC in WT mice (LC3-II/LC3-I ratio: /5.4, P = 0.0159), but preserved in human ?3-AR expressing cells and mice, together with reduced hypertrophy. CONCLUSIONS:Cardiac-specific moderate expression of ?3-AR inhibits the hypertrophic response in part through AMPK activation followed by inhibition of protein synthesis and preservation of autophagy. Activation of the cardiac ?3-AR pathway may provide future therapeutic avenues for the modulation of hypertrophic remodelling.

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

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