Rapamycin-insensitive Regulation of 4E-BP1 in Regenerating Rat Liver

Abstract

In cultured cells, growth factor-induced phosphorylation of two translation modulators, p70 S6 kinase and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), is blocked by nanomolar concentrations of the immunosuppressant rapamycin. Rapamycin also attenuates liver regeneration after partial hepatectomy, but it is not known if this growth-suppressive effect is due to dephosphorylation of p70 S6 kinase and/or 4E-BP1. We found that partial hepatectomy induced a transient increase in liver p70 S6 kinase activity and 4E-BP1 phosphorylation as compared with sham-operated rats. The amount of p70 S6 kinase protein in regenerating liver did not increase, but active kinase from partially hepatectomized animals was highly phosphorylated. Phosphorylated 4E-BP1 from regenerating liver was unable to form an inhibitory complex with initiation factor 4E. Rapamycin blocked the activation of p70 S6 kinase in response to partial hepatectomy in a dose-dependent manner, but 4E-BP1 phosphorylation was not inhibited. By contrast, functional phosphorylation of 4E-BP1 induced by injection of cycloheximide or growth factors was partially reversed by the drug. The mammalian target of rapamycin (mTOR) has been proposed to directly phosphorylate 4E-BP1. Western blot analysis using phospho-specific antibodies showed that phosphorylation of Thr-36/45 and Ser-64 increased in response to partial hepatectomy in a rapamycin-resistant manner. Thus, rapamycin inhibits p70 S6 kinase activation and liver regeneration, but not functional phosphorylation of 4E-BP1, in response to partial hepatectomy. These results indicate that the effect of rapamycin on 4E-BP1 function in vivo can be significantly different from its effect in cultured cells.Most of the cells in the adult mammalian liver are in a quiescent state, but they are induced to re-enter the cell cycle by chemical or surgical treatments that remove or destroy a portion of the liver. A commonly used method for inducing liver regeneration in the rat is by performing a partial hepatectomy in which the two main lobes of the liver are excised, resulting in 68% tissue loss (1). After partial hepatectomy, the remaining liver cells rapidly undergo one or two proliferative cycles so that the mass of the liver remnant nearly doubles after 2 days and approaches the original liver weight by 7 days. The kinetics of this proliferative response have been well documented (reviewed in Ref.2). DNA synthesis in parenchymal cells (hepatocytes) begins about 12 h after partial hepatectomy and reaches a peak by 20 -24 h, followed by a gradual decline. Mitosis follows 6 -8 h later. Nonparenchymal cells lag 24 h behind hepatocytes in initiating DNA synthesis and mitosis. DNA synthesis and restoration of the cell deficit in the liver remnant are mostly complete by 72 h.Partial hepatectomy also leads to an increase in hepatic protein synthesis that is essential for regeneration (3, 4). Superimposed on a general increase in the rate of protein synthesis are marked increases in translation of specific mRNAs (5). Ex...

Repression of Mammalian Target of Rapamycin Complex 1 Inhibits Intestinal Regeneration in Acute Inflammatory Bowel Disease Models

“…1A). This is consistent with the report that mTOR is activated in intestinal ischemia-and irradiation-induced intestinal regeneration (37,38). Inhibition of mTORC1 activity by either rapamycin treatment or haploinsufficiency of Rheb in mice resulted in increased apoptosis and decreased cell proliferation, leading to more severe tissue damage in our DSS-or TBNS-induced colitis models.…”

“…3), suggesting these two key downstream components of mTORC1 play distinct roles in intestinal regeneration. It is reported that rapamycin efficiently inhibits the activity of S6K1, but not 4EBP1 phosphorylation (37). We showed that, in the DSS-induced colitis model, the mice were dead after treatment of rapamycin (Fig.…”

Oleate-mediated activation of phospholipase D and mammalian target of rapamycin (mTOR) regulates proliferation and rapamycin sensitivity of hepatocarcinoma cells

“…Considering that physiologically relevant concentrations of oleate confer rapamycin resistance, one would expect that in obese patients suffering from liver cancer the use of this drug will have limited therapeutic success, unless specific dietary recommendations are implemented. In addition, our finding that oleate activates the mTOR pathway may be relevant to liver regeneration, given that NEFA uptake and mTOR activation are essential to this process [36,37]. Our work begets the clinically important question of whether the proliferative effect of oleate applies to the whole spectrum of obesity-associated cancers, notably those of epithelial origin, or only part of it.…”

Prognostic impact of the activation status of the Akt/mTOR pathway in synovial sarcoma

“…This may correspond to the finding that the immunohistochemically positive ratios of these proteins were relatively low. Another explanation is that they may be activated by kinases other than mTOR 21,22 in skeletal muscle tissue, which was used as a control in the current study. These proteins had lower expression in recurrent or metastatic lesions than in primary tumors according to our Western blot analysis; however, the cause of this phenomenon remains unknown.…”