Mice Deficient in Ribosomal Protein S6 Phosphorylation Suffer from Muscle Weakness that Reflects a Growth Defect and Energy Deficit

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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

Mice deficient in ribosomal protein S6 phosphorylation suffer from muscle weakness that reflects a growth defect and energy deficit. PLoS One

Abstract Background: Mice, whose ribosomal protein S6 cannot be phosphorylated due to replacement of all five phosphorylatable serine residues by alanines (rpS6 P2/2 ), 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 b-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

Phosphorylated Ribosomal Protein S6 Is Required for Akt-Driven Hyperplasia and Malignant Transformation, but Not for Hypertrophy, Aneuploidy and Hyperfunction of Pancreatic β-Cells

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.Fil: Wittenberg, Avigail Dreazen. The Hebrew University Of Jerusalem; IsraelFil: Azar, Shahar. The Hebrew University Of Jerusalem; IsraelFil: Klochendler, Agnes. The Hebrew University Of Jerusalem; IsraelFil: Stolovich-Rain, Miri. The Hebrew University Of Jerusalem; IsraelFil: Avraham, Shlomit. The Hebrew University Of Jerusalem; IsraelFil: Birnbaum, Lea. The Hebrew University Of Jerusalem; IsraelFil: Binder Gallimidi, Adi. The Hebrew University Of Jerusalem; IsraelFil: Katz, Maximiliano Javier. The Hebrew University Of Jerusalem; Israel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Dor, Yuval. The Hebrew University Of Jerusalem; IsraelFil: Meyuhas, Oded. The Hebrew University Of Jerusalem; Israe

The amount of mitochondria and the activity of complexes of the oxidative phosphorylation system are unchanged in rpS6^P−/− muscle.

<p>Mitochondria were isolated from hind limb muscle of four age-matched rpS6^P+/+ (WT) and rpS6^P−/− (−/−) male mice and were assayed for citrate synthase (A), cytochrome C oxidase (B), as well as complexes I (C), II (D), II+III (E) and IV (F) of the oxidative phosphorylation system. The activity of the different complexes was normalized to that of citrate synthase and the results are presented as relative activities. Vertical bars represent SEM (n = 4 age- matched male mice for each genotype).</p

Insulin-induced signaling and glucose uptake are similar in rpS6^P−/− and rpS6^p+/+ muscles.

<p>(A) Soleus muscles were excised from rpS6^P−/− (−/−) and rpS6^p+/+ (WT) 2- to 3-mo-old male mice following 16 h starvation and intraperitoneal injection of saline (−) or 2.5 U of insulin/kg of body weight (+) for 5 min. Cytoplasmic extracts were subjected to Western blot analysis with the indicated antibodies. Note the enrichment for the upper band of 4E-BP1 (hyperphosphorylated form) upon insulin treatment. (B) Insulin-induced uptake of glucose into isolated muscle. Right and left soleus muscles were isolated from the hind limbs of 16 h starved rpS6^P−/− (−/−) and rpS6^p+/+ (WT) male mice. 2-Deoxy glucose uptake is presented as µmol per gram tissue per h. The data are presented as average±SEM for 5 rpS6^P−/− and 4 S6^p+/+ age-matched (7–9 weeks) male mice. (C) Mice were injected intraperitoneally with 0.25 U insulin/kg body weight and within seconds with 10–24 Ci [¹⁸F]fluoro-2-deoxyglucose (FDG) into the tail vein. The radioactivity concentration in hind limb muscles was estimated at 40 to 45 min after FDG injection by positron emission tomography. The concentration of radioactivity is presented as % injected dose (ID) per gm body weight per ml tissue. The data are shown as average±SEM for 5 male mice of each genotype.</p

Newly formed myofibers are smaller in a rpS6^P−/− muscle.

<p>(A) Sections of tibialis anterior of 2-mo-old rpS6^P+/+ and rpS6^P−/− mice were prepared 5 days after cryoinjury, and were immunostained for eMHC. One representative section is presented. (B) Enlargement of the framed area in (A). (C) Digital images of the immunostained sections were used for measurement of CSA of individual eMHC-positive myofibers, as described in “Experimental Procedures”. The average CSA of rpS6^P−/− myofibers (n = 327) was normalized to that of their wild type counterparts (n = 210), which was arbitrarily set at 1. The results are presented as average±SEM. *, p<0.0001.</p

Stores of glycogen and triacylglycerol are larger and similar, respectively, in rpS6^P−/− muscle.

<p>(A) Soleus glycogen content. Soleus muscles were excised from 20 rpS6^P+/+ (WT) and 15 age-matched rpS6^P−/− (−/−) male mice and their glycogen content was measured. (B) Glycogen synthase (GS) and (C) glycogen phosphorylase (GP) activities were assayed in the same extracts (n = 4 age-matched male mice for each genotype). (D) and (E) TG content in soleus and gastrocnemious, respectively. 5 soleus (left and right muscles were pooled) and 10 individual gastrocnemious muscles were excised from 5 rpS6^P+/+ (WT) and 5 age-matched rpS6^P−/− (−/−) male mice and their TG content was measured. All results are presented as average±SEM.</p

Muscle strength is impaired in rpS6^P−/− mice.

<p>(A to C) Age-matched (4 and 7 month) male mice (n = 13 for WT and 12 for rpS6^P−/− mice) were subjected to screening, according to the SHIRPA behavioral protocol (see details in “Experimental Procedures”). (A) Grip strength. Mice were allowed to grip a grid and a gentle horizontal backwards pull through their tail was applied. Higher scores indicate greater grip strength. An unbiased observer, blinded to the genotype, performed the experiment in a blind fashion; (B) Wire maneuver. Results represent time in seconds required for a mouse that is hung from a wire with its forearms to elevate its hind limbs and grip the wire. (C) Rota-rod performance. Mice were placed on a moving cylinder, which was gradually accelerated from an initial speed of 4 rpm to a maximum of 40 rpm. Latency to fall from the rota-rod is presented in seconds. Motor performance was measured in three 10 min sessions (time 0, 1 h and 24 h). In each trial, the time in seconds until falling off was recorded. *<i>P</i><0.0005 for each trial versus rpS6^P+/+ mice. (D) Endurance test. 5 rpS6^p+/+ and 4 S6^P−/− age-matched (7–9 weeks) male mice were allowed to run on the treadmill set with a slope of 12.5 degree and a speed of 20 m/min. The results represent the total running time with two attempts to pause. Results of all experiments are presented as average±SEM, and the absence of SEM bars for some measurements simply reflects a value close to zero that is graphically invisible.</p

rpS6 phosphorylation deficiency renders Ras^G12D MEFs more sensitive to both genotoxic and proteotoxic stresses.

<p>(A) Immortalized WT, rpS6^P-/-, Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (B) Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were seeded in 96-well plates at a density of 4 x10³ per well. Proliferation was monitored by measuring the A₆₅₀ of the methylene-blue dye extracted from stained cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149995#pone.0149995.ref032" target="_blank">32</a>]. Absorbance measured 24 h after platting, was set arbitrarily at 1 and absorbance measured at later time points (average ± SEM [n = 6]) for each time point) was normalized to that value. t_d, population-doubling time. (C) Immortalized Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were incubated with the indicated concentrations of etoposide for 24 h, harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (D) Immortalized Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were incubated with the indicated concentrations of MG132 for 24 h, harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies.</p

Constitutive expression of Akt induces polyploidy regardless of the rpS6 phosphorylation status.

<p>A. rpS6 deficiency does not affect ploidy in β-cells. Islets were isolated from wild-type (WT) and 2 month-old Akt^tg mice. Dissociated islets were fixed, permeabilized, immunostained for Insulin, HA, Ki67 and Hoechst before FACS analysis. The DNA content of 5,000 to 10,000 quiescent β-cells (insulin-positive, Ki67-negative) was measured by FACS analysis. B. 5,000 quiescent (Ki67-negative) β-cells (insulin-positive) that express myr-Akt (HA-positive) are polyploidy, whereas their HA-negative counterpart shows mostly a diploid profile as wild-type β-cells. C. rpS6 deficiency does not affect the ploidy profile of myr-Akt expressing β-cells. Islets were isolated from 2 month-old mice (three wild-type (WT), two Akt^tg and three rpS6^P-/-;Akt^tg mice) and 5,000 to 10,000 HA-positive β-cells processed as in (A) for FACS analysis.</p