IGF-I, EGF, and sex steroids regulate autophagy in bovine mammary epithelial cells via the mTOR pathway.
ABSTRACT Mammary gland growth and involution are based on a dynamic equilibrium between proliferation and apoptosis of mammary epithelial cells (MEC). The main type of cell death responsible for bovine mammary gland involution is apoptosis, but MEC also exhibit morphological features of autophagy. The present study has been undertaken in order to examine factors, which are responsible for the regulation of autophagy in bovine MEC. We used a model of in vitro mammary gland involution known to be dependent on fetal bovine serum (FBS) deficiency in the culture of bovine BME-UV1 cells. We investigated the effects of insulin-like growth factor-1 (IGF-I) and epidermal growth factor (EGF) signaling, as well as sex steroids and rapamycin (a specific inhibitor of mammalian target of rapamycin, mTOR, kinase) on autophagy in the MEC line BME-UV1. Our main focus was on the role of mTOR in the regulation of autophagy by growth factors and hormones. Laser scanning cytometry, electron microscopy, Western-blot analysis, GFP-LC3 reporter-based expression analysis, and LysoTracker Green-related fluorescence were used to determine the activity of autophagy in BME-UV1 cells. We found that FBS deficiency induced both autophagy and apoptosis with the highest intensity of both processes after 48h of MEC exposure to the deficient medium (0.5% FBS). Addition of IGF-I or/and EGF to the FBS-deficient medium clearly diminished autophagy. We also show that IGF-I and EGF are involved in the activation of mTOR in bovine MEC, whereas inhibition of mTOR by rapamycin abrogated the suppressive effects of IGF-I and EGF on autophagy. This suggests that mTOR links IGF-I and EGF signaling in inhibiting the autophagy pathways. Contrary to IGF-I and EGF, 17beta-estradiol and progesterone exerted stimulatory effects on autophagy in bovine MEC. At the same time we observed a suppressive effect of both steroids on mTOR activation/phosphorylation. In conclusion, autophagy in bovine MEC undergoes complex regulation, where its activity is controlled by survival pathways dependent on IGF-I and EGF, which are involved in suppression of autophagy, and by pregnancy steroids, which act as inducers of the process.
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ABSTRACT: Insulin-like growth factor-1 (IGF-1) signaling is a key pathway in the control of cell growth and survival. Three critical nodes in the IGF-1 signaling pathway have been described in cardiomyo-cytes: protein kinase Akt/mammalian target of rapamycin (mTOR), Ras/Raf/extracellular signal-regulated kinase (ERK), and phospholipase C (PLC)/inositol 1,4,5-triphosphate (InsP 3)/ Ca 21 . The Akt/mTOR and Ras/Raf/ERK signaling arms govern survival in the settings of cardiac stress and hypertrophic growth. By contrast, PLC/InsP 3 /Ca 21 functions to regulate meta-bolic adaptability and gene transcription. Autophagy is a cata-bolic process involved in protein degradation, organelle turnover, and nonselective breakdown of cytoplasmic compo-nents during nutrient starvation or stress. In the heart, autoph-agy is observed in a variety of human pathologies, where it can be either adaptive or maladaptive, depending on the context. We proposed the hypothesis that IGF-1 protects the heart by res-cuing the mitochondrial metabolism and the energetics state, reducing cell death and controls the potentially exacerbate auto-phagic response to nutritional stress. In light of the importance of IGF-1 and autophagy in the heart, we review here IGF-1 sig-naling and autophagy regulation in the context of cardiomyo-cyte nutritional stress. V C 2013 IUBMB Life, 00(00):000–000, 2013.
Article: IGF-1 stimulates protein synthesis by enhanced signaling through mTORC1 in bovine mammary epithelial cells.[show abstract] [hide abstract]
ABSTRACT: Using the MAC-T cell line as a model, the effects of insulin-like growth factor (IGF)-1 on the regulation of protein synthesis through the mammalian target of rapamycin complex 1 (mTORC1) signaling in bovine mammary epithelial cells were evaluated. Global rates of protein synthesis increased by 47% within 30 min of IGF-1 treatment. The effect of IGF-1 on protein synthesis was associated with enhanced association of the eukaryotic initiation factor (eIF) 4E with eIF4G and a concomitant reduction of eIF4E association with eIF4E-binding protein-1 (4E-BP1). There was a progressive increase in the phosphorylation state of ribosomal protein S6 kinase-1, a downstream target of mTORC1 in response to IGF-1. In addition, IGF-1 stimulated mTORC1 kinase activity toward 4E-BP1 in vitro. Phosphorylation on Ser473 of Akt was induced by IGF-1 within 5 min and remained elevated throughout a 30-min time course. The effect of IGF-1 on Akt phosphorylation was also concentration dependent. Activation of Akt by IGF-1 led to increased phosphorylation of tuberous sclerosis complex 2 on Thr1426, without any change in its association with tuberous sclerosis complex 1. Phosphorylation of proline-rich Akt substrate of 40-kDa (PRAS40) at Thr246 was stimulated by IGF-1. The amount of PRAS40 associated with mTORC1 decreased in response to IGF-1, and PRAS40 binding to mTORC1 was inversely related to its phosphorylation level. Overall, these results suggest that activation of the PI3K-Akt pathway by IGF-1 stimulated global protein synthesis in bovine mammary epithelial cells through changes in the phosphorylation and association state of components of the mTORC1 signaling pathway.Domestic animal endocrinology 11/2009; 38(4):211-21. · 1.65 Impact Factor
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ABSTRACT: Insulin-like growth factor 1 (IGF-1) is known to exert cardioprotective actions. However, it remains unknown if autophagy, a major adaptive response to nutritional stress, contributes to IGF-1-mediated cardioprotection. We subjected cultured neonatal rat cardiomyocytes, as well as live mice, to nutritional stress and assessed cell death and autophagic rates. Nutritional stress induced by serum/glucose deprivation strongly induced autophagy and cell death, and both responses were inhibited by IGF-1. The Akt/mammalian target of rapamycin (mTOR) pathway mediated the effects of IGF-1 upon autophagy. Importantly, starvation also decreased intracellular ATP levels and oxygen consumption leading to AMP-activated protein kinase (AMPK) activation; IGF-1 increased mitochondrial Ca(2+) uptake and mitochondrial respiration in nutrient-starved cells. IGF-1 also rescued ATP levels, reduced AMPK phosphorylation and increased p70(S6K) phosphorylation, which indicates that in addition to Akt/mTOR, IGF-1 inhibits autophagy by the AMPK/mTOR axis. In mice harbouring a liver-specific igf1 deletion, which dramatically reduces IGF-1 plasma levels, AMPK activity and autophagy were increased, and significant heart weight loss was observed in comparison with wild-type starved animals, revealing the importance of IGF-1 in maintaining cardiac adaptability to nutritional insults in vivo. Our data support the cardioprotective actions of IGF-1, which, by rescuing the mitochondrial metabolism and the energetic state of cells, reduces cell death and controls the potentially harmful autophagic response to nutritional challenges. IGF-1, therefore, may prove beneficial to mitigate damage induced by excessive nutrient-related stress, including ischaemic disease in multiple tissues.Cardiovascular research 12/2011; 93(2):320-9. · 5.80 Impact Factor