Secondary hyperparathyroidism is characterized by increased serum parathyroid hormone (PTH) level and parathyroid cell proliferation. However, the molecular pathways mediating the increased ...parathyroid cell proliferation remain undefined. Here, we found that the mTOR pathway was activated in the parathyroid of rats with secondary hyperparathyroidism induced by either chronic hypocalcemia or uremia, which was measured by increased phosphorylation of ribosomal protein S6 (rpS6), a downstream target of the mTOR pathway. This activation correlated with increased parathyroid cell proliferation. Inhibition of mTOR complex 1 by rapamycin decreased or prevented parathyroid cell proliferation in secondary hyperparathyroidism rats and in vitro in uremic rat parathyroid glands in organ culture. Knockin rpS6(p-/-) mice, in which rpS6 cannot be phosphorylated because of substitution of all five phosphorylatable serines with alanines, had impaired PTH secretion after experimental uremia- or folic acid-induced AKI. Uremic rpS6(p-/-) mice had no increase in parathyroid cell proliferation compared with a marked increase in uremic wild-type mice. These results underscore the importance of mTOR activation and rpS6 phosphorylation for the pathogenesis of secondary hyperparathyroidism and indicate that mTORC1 is a significant regulator of parathyroid cell proliferation through rpS6.
The signaling pathways that mediate the development of pancreatic ductal adenocarcinoma (PDAC) downstream of mutant Kras remain incompletely understood. Here, we focus on ribosomal protein S6 (rpS6), ...an mTOR effector not implicated previously in cancer. Phosphorylation of rpS6 was increased in pancreatic acinar cells upon implantation of the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) or transgenic expression of mutant Kras. To examine the functional significance of rpS6 phosphorylation, we used knockin mice lacking all five phosphorylatable sites in rpS6 (termed rpS6(P-/-) mice). Strikingly, the development of pancreatic cancer precursor lesions induced by either DMBA or mutant Kras was greatly reduced in rpS6(P-/-) mice. The rpS6 mutants expressing oncogenic Kras showed increased p53 along with increased staining of γ-H2AX and 53bp1 (Trp53bp1) in areas of acinar ductal metaplasia, suggesting that rpS6 phosphorylation attenuates Kras-induced DNA damage and p53-mediated tumor suppression. These results reveal that rpS6 phosphorylation is important for the initiation of pancreatic cancer.
Constitutive expression of active Akt (Akttg) drives hyperplasia and hypertrophy of pancreatic β-cells, concomitantly with increased insulin secretion and improved glucose tolerance, and at a later ...stage the development of insulinoma. To determine which functions of Akt are mediated by ribosomal protein S6 (rpS6), an Akt effector, we generated mice that express constitutive Akt in β-cells in the background of unphosphorylatable ribosomal protein S6 (rpS6P-/-). rpS6 phosphorylation deficiency failed to block Akttg-induced hypertrophy and aneuploidy in β-cells, as well as the improved glucose homeostasis, indicating that Akt carries out these functions independently of rpS6 phosphorylation. In contrast, rpS6 phosphorylation deficiency efficiently restrained the reduction in nuclear localization of the cell cycle inhibitor p27, as well as the development of Akttg-driven hyperplasia and tumor formation in β-cells. In vitro experiments with Akttg and rpS6P-/-;Akttg fibroblasts demonstrated that rpS6 phosphorylation deficiency leads to reduced translation fidelity, which might underlie its anti-tumorigenic effect in the pancreas. However, the role of translation infidelity in tumor suppression cannot simply be inferred from this heterologous experimental model, as rpS6 phosphorylation deficiency unexpectedly elevated the resistance of Akttg fibroblasts to proteotoxic, genotoxic as well as autophagic stresses. In contrast, rpS6P-/- fibroblasts exhibited a higher sensitivity to these stresses upon constitutive expression of oncogenic Kras. The latter result provides a possible mechanistic explanation for the ability of rpS6 phosphorylation deficiency to enhance DNA damage and protect mice from Kras-induced neoplastic transformation in the exocrine pancreas. We propose that Akt1 and Kras exert their oncogenic properties through distinct mechanisms, even though both show addiction to rpS6 phosphorylation.
Mice, whose ribosomal protein S6 cannot be phosphorylated due to replacement of all five phosphorylatable serine residues by alanines (rpS6(P-/-)), are viable and fertile. However, phenotypic ...characterization of these mice and embryo fibroblasts derived from them, has established the role of these modifications in the regulation of the size of several cell types, as well as pancreatic beta-cell function and glucose homeostasis. A relatively passive behavior of these mice has raised the possibility that they suffer from muscle weakness, which has, indeed, been confirmed by a variety of physical performance tests.
A large variety of experimental methodologies, including morphometric measurements of histological preparations, high throughput proteomic analysis, positron emission tomography (PET) and numerous biochemical assays, were used in an attempt to establish the mechanism underlying the relative weakness of rpS6(P-/-) muscles. Collectively, these experiments have demonstrated that the physical inferiority appears to result from two defects: a) a decrease in total muscle mass that reflects impaired growth, rather than aberrant differentiation of myofibers, as well as a diminished abundance of contractile proteins; and b) a reduced content of ATP and phosphocreatine, two readily available energy sources. The abundance of three mitochondrial proteins has been shown to diminish in the knockin mouse. However, the apparent energy deficiency in this genotype does not result from a lower mitochondrial mass or compromised activity of enzymes of the oxidative phosphorylation, nor does it reflect a decline in insulin-dependent glucose uptake, or diminution in storage of glycogen or triacylglycerol (TG) in the muscle.
This study establishes rpS6 phosphorylation as a determinant of muscle strength through its role in regulation of myofiber growth and energy content. Interestingly, a similar role has been assigned for ribosomal protein S6 kinase 1, even though it regulates myoblast growth in an rpS6 phosphorylation-independent fashion.
Ribosomal protein S6 kinase (S6K) has been implicated in the phosphorylation of multiple substrates and is subject to activation by a wide variety of signals that converge at mammalian target of ...rapamycin (mTOR). In the course of the search for its physiological role, it was proposed that S6K activation and ribosomal protein S6 (rpS6) phosphorylation account for the translational activation of a subgroup of transcripts, the TOP mRNAs. The structural hallmark of these mRNAs is an oligopyrimidine tract at their 5'-terminus, known as the 5'-TOP motif. TOP mRNAs consists of about 90 members that encode multiple components of the translational machinery, such as ribosomal proteins and translation factors. The translation efficiency of TOP mRNAs indeed correlates with S6K activation and rpS6 phosphorylation, yet recent biochemical and genetic studies have established that, although S6K and TOP mRNAs respond to similar signals and are regulated by mTOR, they maintain no cause and effect relationship. Instead, S6K is primarily involved in regulation of cell size, and affects glucose homeostasis, but is dispensable for global protein synthesis, whereas translational efficiency of TOP mRNAs is a determinant of the cellular protein synthesis capacity. Despite extensive studies of their function and mode of regulation, the mechanism underlying the effect of S6K on the cell size, as well as the trans-acting factor that mediates the translational control of TOP mRNAs, still await their identification.
Constitutive expression of active Akt (Akt.sup.tg) drives hyperplasia and hypertrophy of pancreatic beta-cells, concomitantly with increased insulin secretion and improved glucose tolerance, and at a ...later stage the development of insulinoma. To determine which functions of Akt are mediated by ribosomal protein S6 (rpS6), an Akt effector, we generated mice that express constitutive Akt in beta-cells in the background of unphosphorylatable ribosomal protein S6 (rpS6.sup.P-/-). rpS6 phosphorylation deficiency failed to block Akt.sup.tg -induced hypertrophy and aneuploidy in beta-cells, as well as the improved glucose homeostasis, indicating that Akt carries out these functions independently of rpS6 phosphorylation. In contrast, rpS6 phosphorylation deficiency efficiently restrained the reduction in nuclear localization of the cell cycle inhibitor p27, as well as the development of Akt.sup.tg -driven hyperplasia and tumor formation in beta-cells. In vitro experiments with Akt.sup.tg and rpS6.sup.P-/- ;Akt.sup.tg fibroblasts demonstrated that rpS6 phosphorylation deficiency leads to reduced translation fidelity, which might underlie its anti-tumorigenic effect in the pancreas. However, the role of translation infidelity in tumor suppression cannot simply be inferred from this heterologous experimental model, as rpS6 phosphorylation deficiency unexpectedly elevated the resistance of Akt.sup.tg fibroblasts to proteotoxic, genotoxic as well as autophagic stresses. In contrast, rpS6.sup.P-/- fibroblasts exhibited a higher sensitivity to these stresses upon constitutive expression of oncogenic Kras. The latter result provides a possible mechanistic explanation for the ability of rpS6 phosphorylation deficiency to enhance DNA damage and protect mice from Kras-induced neoplastic transformation in the exocrine pancreas. We propose that Akt1 and Kras exert their oncogenic properties through distinct mechanisms, even though both show addiction to rpS6 phosphorylation.
Constitutive expression of active Akt (Akttg) drives hyperplasia and hypertrophy of pancreatic Beta-cells, concomitantly with increased insulin secretion and improved glucose tolerance, and at a ...later stage the development of insulinoma. To determine which functions of Akt are mediated by ribosomal protein S6 (rpS6), an Akt effector, we generated mice that express constitutive Akt in Beta-cells in the background of unphosphorylatable ribosomal protein S6 (rpS6P-/-). rpS6 phosphorylation deficiency failed to block Akttg-induced hypertrophy and aneuploidy in Beta-cells, as well as the improved glucose homeostasis, indicating that Akt carries out these functions independently of rpS6 phosphorylation. In contrast, rpS6 phosphorylation deficiency efficiently restrained the reduction in nuclear localization of the cell cycle inhibitor p27, as well as the development of Akttg-driven hyperplasia and tumor formation in Beta-cells. In vitro experiments with Akttg and rpS6P-/-; Akttg fibroblasts demonstrated that rpS6 phosphorylation deficiency leads to reduced translation fidelity, which might underlie its anti-tumorigenic effect in the pancreas. However, the role of translation infidelity in tumor suppression cannot simply be inferred from this heterologous experimental model, as rpS6 phosphorylation deficiency unexpectedly elevated the resistance of Akttg fibroblasts to proteotoxic, genotoxic as well as autophagic stresses. In contrast, rpS6P-/- fibroblasts exhibited a higher sensitivity to these stresses upon constitutive expression of oncogenic Kras. The latter result provides a possible mechanistic explanation for the ability of rpS6 phosphorylation deficiency to enhance DNA damage and protect mice from Kras-induced neoplastic transformation in the exocrine pancreas. We propose that Akt1 and Kras exert their oncogenic properties through distinct mechanisms, even though both show addiction to rpS6 phosphorylation.
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