Mario Pende

S6K1−/−/S6K2−/− Mice Exhibit Perinatal Lethality and Rapamycin-Sensitive 5′-Terminal Oligopyrimidine mRNA Translation and Reveal a Mitogen-Activated Protein Kinase-Dependent S6 Kinase Pathway

ABSTRACT Activation of 40S ribosomal protein S6 kinases (S6Ks) is mediated by anabolic signals triggered by hormones, growth factors, and nutrients. Stimulation by any of these agents is inhibited by the bacterial macrolide rapamycin, which binds to and inactivates the mammalian target of rapamycin, an S6K kinase. In mammals, two genes encoding homologous S6Ks, S6K1 and S6K2, have been identified. Here we show that mice deficient for S6K1 or S6K2 are born at the expected Mendelian ratio. Compared to wild-type mice, S6K1−/− mice are significantly smaller, whereas S6K2 −/− mice tend to be slightly larger. However, mice lacking both genes showed a sharp reduction in viability due to perinatal lethality. Analysis of S6 phosphorylation in the cytoplasm and nucleoli of cells derived from the distinct S6K genotypes suggests that both kinases are required for full S6 phosphorylation but that S6K2 may be more prevalent in contributing to this response. Despite the impairment of S6 phosphorylation in cells from S6K1 −/−/S6K2 −/− mice, cell cycle progression and the translation of 5′-terminal oligopyrimidine mRNAs were still modulated by mitogens in a rapamycin-dependent manner. Thus, the absence of S6K1 and S6K2 profoundly impairs animal viability but does not seem to affect the proliferative responses of these cell types. Unexpectedly, in S6K1 −/−/S6K2 −/− cells, S6 phosphorylation persisted at serines 235 and 236, the first two sites phosphorylated in response to mitogens. In these cells, as well as in rapamycin-treated wild-type, S6K1 −/−, and S6K2 −/− cells, this step was catalyzed by a mitogen-activated protein kinase (MAPK)-dependent kinase, most likely p90rsk. These data reveal a redundancy between the S6K and the MAPK pathways in mediating early S6 phosphorylation in response to mitogens.

Disruption of the p70 s6k /p85 s6k gene reveals a small mouse phenotype and a new functional S6 kinase

Recent studies have shown that the p70s6k/p85s6k signaling pathway plays a critical role in cell growth by modulating the translation of a family of mRNAs termed 5′TOPs, which encode components of the protein synthetic apparatus. Here we demonstrate that homozygous disruption of the p70s6k/p85s6k gene does not affect viability or fertility of mice, but that it has a significant effect on animal growth, especially during embryogenesis. Surprisingly, S6 phosphorylation in liver or in fibroblasts from p70s6k/p85s6k‐deficient mice proceeds normally in response to mitogen stimulation. Furthermore, serum‐induced S6 phosphorylation and translational up‐regulation of 5′TOP mRNAs were equally sensitive to the inhibitory effects of rapamycin in mouse embryo fibroblasts derived from p70s6k/p85s6k‐deficient and wild‐type mice. A search of public databases identified a novel p70s6k/p85s6k homolog which contains the same regulatory motifs and phosphorylation sites known to control kinase activity. This newly identified gene product, termed S6K2, is ubiquitously expressed and displays both mitogen‐dependent and rapamycin‐sensitive S6 kinase activity. More striking, in p70s6k/p85s6k‐deficient mice, the S6K2 gene is up‐regulated in all tissues examined, especially in thymus, a main target of rapamycin action. The finding of a new S6 kinase gene, which can partly compensate for p70s6k/p85s6k function, underscores the importance of S6K function in cell growth.

Hypoinsulinaemia, glucose intolerance and diminished beta-cell size in S6K1-deficient mice

Insulin controls glucose homeostasis by regulating glucose use in peripheral tissues, and its own production and secretion in pancreatic β cells. These responses are largely mediated downstream of the insulin receptor substrates, IRS-1 and IRS-2 (refs 4,5,6,7,8), through distinct signalling pathways. Although a number of effectors of these pathways have been identified, their roles in mediating glucose homeostasis are poorly defined. Here we show that mice deficient for S6 kinase 1, an effector of the phosphatidylinositide-3-OH kinase signalling pathway, are hypoinsulinaemic and glucose intolerant. Whereas insulin resistance is not observed in isolated muscle, such mice exhibit a sharp reduction in glucose-induced insulin secretion and in pancreatic insulin content. This is not due to a lesion in glucose sensing or insulin production, but to a reduction in pancreatic endocrine mass, which is accounted for by a selective decrease in β-cell size. The observed phenotype closely parallels those of preclinical type 2 diabetes mellitus, in which malnutrition-induced hypoinsulinaemia predisposes individuals to glucose intolerance.

The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity

The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin‐sensitive manner. Here we demonstrate that the p90 ribosomal protein S6 kinase (RSK) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor‐dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3.

Atrophy of S6K1-/- skeletal muscle cells reveals distinct mTOR effectors for cell cycle and size control

The mammalian target of rapamycin (mTOR) and Akt proteins regulate various steps of muscle development and growth, but the physiological relevance and the downstream effectors are under investigation. Here we show that S6 kinase 1 (S6K1), a protein kinase activated by nutrients and insulin-like growth factors (IGFs), is essential for the control of muscle cytoplasmic volume by Akt and mTOR. Deletion of S6K1 does not affect myoblast cell proliferation but reduces myoblast size to the same extent as that observed with mTOR inhibition by rapamycin. In the differentiated state, S6K1−/− myotubes have a normal number of nuclei but are smaller, and their hypertrophic response to IGF1, nutrients and membrane-targeted Akt is blunted. These growth defects reveal that mTOR requires distinct effectors for the control of muscle cell cycle and size, potentially opening new avenues of therapeutic intervention against neoplasia or muscle atrophy.

Gluco-incretins control insulin secretion at multiple levels as revealed in mice lacking GLP-1 and GIP receptors

The role of the gluco-incretin hormones GIP and GLP-1 in the control of beta cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both. Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated. After intraperitoneal injections, glucose intolerance was more severe in double- as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion. When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R(-/-) but not in Gipr(-/-) mice. In perifusion experiments, however, first-phase insulin secretion was present in both types of islets. In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization. Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on beta cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a beta cell autonomous secretion defect when both receptors are inactivated.

AKT2 is essential to maintain podocyte viability and function during chronic kidney disease

In chronic kidney disease (CKD), loss of functional nephrons results in metabolic and mechanical stress in the remaining ones, resulting in further nephron loss. Here we show that Akt2 activation has an essential role in podocyte protection after nephron reduction. Glomerulosclerosis and albuminuria were substantially worsened in Akt2−/− but not in Akt1−/− mice as compared to wild-type mice. Specific deletion of Akt2 or its regulator Rictor in podocytes revealed that Akt2 has an intrinsic function in podocytes. Mechanistically, Akt2 triggers a compensatory program that involves mouse double minute 2 homolog (Mdm2), glycogen synthase kinase 3 (Gsk3) and Rac1. The defective activation of this pathway after nephron reduction leads to apoptosis and foot process effacement of the podocytes. We further show that AKT2 activation by mammalian target of rapamycin complex 2 (mTORC2) is also required for podocyte survival in human CKD. More notably, we elucidate the events underlying the adverse renal effect of sirolimus and provide a criterion for the rational use of this drug. Thus, our results disclose a new function of Akt2 and identify a potential therapeutic target for preserving glomerular function in CKD.

mTORC1 mediated translational elongation limits intestinal tumour initiation and growth

Inactivation of APC is a strongly predisposing event in the development of colorectal cancer, prompting the search for vulnerabilities specific to cells that have lost APC function. Signalling through the mTOR pathway is known to be required for epithelial cell proliferation and tumour growth, and the current paradigm suggests that a critical function of mTOR activity is to upregulate translational initiation through phosphorylation of 4EBP1 (refs 6, 7). This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 (ref. 8), would be ineffective in limiting cancer progression in APC-deficient lesions. Here we show in mice that mTOR complex 1 (mTORC1) activity is absolutely required for the proliferation of Apc-deficient (but not wild-type) enterocytes, revealing an unexpected opportunity for therapeutic intervention. Although APC-deficient cells show the expected increases in protein synthesis, our study reveals that it is translation elongation, and not initiation, which is the rate-limiting component. Mechanistically, mTORC1-mediated inhibition of eEF2 kinase is required for the proliferation of APC-deficient cells. Importantly, treatment of established APC-deficient adenomas with rapamycin (which can target eEF2 through the mTORC1–S6K–eEF2K axis) causes tumour cells to undergo growth arrest and differentiation. Taken together, our data suggest that inhibition of translation elongation using existing, clinically approved drugs, such as the rapalogs, would provide clear therapeutic benefit for patients at high risk of developing colorectal cancer.

Deletion of ribosomal S6 kinases does not attenuate pathological, physiological, or insulin-like growth factor 1 receptor-phosphoinositide 3-kinase-induced cardiac hypertrophy.

ABSTRACT Ribosomal S6 kinases (S6Ks) have been depicted as critical effectors downstream of growth factor pathways, which play an important role in the regulation of protein synthesis by phosphorylating the ribosomal protein, S6. The goal of this study was to determine whether S6Ks regulate heart size, are critical for the induction of cardiac hypertrophy in response to a pathological or physiological stimulus, and whether S6Ks are critical downstream effectors of the insulin-like growth factor 1 (IGF1)-phosphoinositide 3-kinase (PI3K) pathway. For this purpose, we generated and characterized cardiac-specific S6K1 and S6K2 transgenic mice and subjected S6K1−/−, S6K2−/−, and S6K1−/− S6K2−/− mice to a pathological stress (aortic banding) or a physiological stress (exercise training). To determine the genetic relationship between S6Ks and the IGF1-PI3K pathway, S6K transgenic and knockout mice were crossed with cardiac-specific transgenic mice overexpressing the IGF1 receptor (IGF1R) or PI3K mutants. Here we show that overexpression of S6K1 induced a modest degree of hypertrophy, whereas overexpression of S6K2 resulted in no obvious cardiac phenotype. Unexpectedly, deletion of S6K1 and S6K2 had no impact on the development of pathological, physiological, or IGF1R-PI3K-induced cardiac hypertrophy. These studies suggest that S6Ks alone are not essential for the development of cardiac hypertrophy.

Important role for AMPKα1 in limiting skeletal muscle cell hypertrophy

Activation of AMP‐activated protein kinase (AMPK) inhibits protein synthesis through the suppression of the mammalian target of rapamycin complex 1 (mTORCl), a critical regulator of muscle growth. The purpose of this investigation was to determine the role of the AMPKα1 catalytic subunit on muscle cell size control and adaptation to muscle hypertrophy. We found that AMPKα1(—/—) primary cultured myotubes and myofibers exhibit larger cell size compared with control cells in response to chronic Akt activation. We next subjected the plantaris muscle of AMPKα1(—/—) and control mice to mechanical overloading to induce muscle hypertrophy. We observed significant elevations of AMPKαl activity in the control muscle at days 7 and 21 after the overload. Overloading‐induced muscle hypertrophy was significantly accelerated in AMPKα1(—/—) mice than in control mice [+32 vs. +53% at day 7 and +57 vs. +76% at day 21 in control vs. AMPKα1(—/—) mice, respectively]. This enhanced growth of AMPKα1‐deficient muscle was accompanied by increased phosphorylation of mTOR signaling downstream targets and decreased phosphorylation of eukaryotic elongation factor 2. These results demonstrate that AMPKα1 plays an important role in limiting skeletal muscle overgrowth during hypertrophy through inhibition of the mTOR‐signaling pathway.—Mounier, R., Louise Lantier, Leclerc, J., Sotiropoulos, A., Pende, M., Daegelen, D., Sakamoto, K., Foretz, M., Viollet, B. Important role for AMPKa1 in limiting skeletal muscle cell hypertrophy. FASEBJ. 23, 2264–2273 (2009)