A High Inorganic Phosphate Diet Perturbs Brain Growth, Alters Akt-ERK
http://www.100md.com
《毒物学科学杂志》
Laboratory of Toxicology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Seoul 151–742, Korea
Department of Food and Nutrition, College of Human Ecology, Seoul National University, Seoul 151–742, Korea
Laboratory of Molecular Oncology, Korea Institute of Radiological & Medical Sciences, Seoul 139–240, Korea
Institut National de la Sante et de la Recherche Medicale U589, Institut Louis Bugnard, IFR31, CHU Rangueil, Batiment L3, Avenue Jean Poulhes, BP 84225, 31432 Toulouse Cedex 4, France
Centre de Biologie du Developpement, CNRS-UMR5547, IFR 109, Universite Paul Sabatier, 31062 Toulouse, France
Emory University School of Medicine, Division of Endocrinology, Metabolism and Lipids, Atlanta GA 30322, USA
ABSTRACT
Inorganic phosphate (Pi) plays a key role in diverse physiological functions. Recently, considerable progress has been made in our understanding of the function and regulation of the brain-specific sodium-dependent inorganic phosphate transporter 1 (NPT1), which is found to exist principally in cerebrum and cerebellum. The potential importance of Pi as a novel signaling molecule and the poor prognosis of diverse neurodegenerative diseases that involve brain-specific NPT1 have prompted us to define the pathways by which Pi affects mouse brain growth. A high phosphate diet caused an increase in serum Pi accompanied by a decrease in calcium, and a decrease in body weight coupled with a decreased relative weight of cerebellum. A high phosphate diet caused a significant increase in protein expression of NPT1, both in cerebrum and cerebellum. Additionally, the high phosphate diet increased Homo sapiens v-akt murine thymoma viral oncogene homolog 1 (Akt) phosphorylation at Ser473 in cerebrum and cerebellum, whereas suppression of Akt phosphorylation at Thr308 was observed only in cerebellum. Selective suppression of eukaryotic translation initiation factor-binding protein (eIF4E-BP1) in cerebrum was induced by high levels of Pi, which induced cap-dependent and cap-independent protein translation in cerebrum and cerebellum, respectively. Phosphorylation of extracellular regulated kinase 1 (ERK1) in comparison with that of ERK2 was significantly reduced in both cerebrum and cerebellum. High levels of Pi reduced protein expressions of proliferating cell nuclear antigen (PCNA) and cyclin D1 in cerebrum and cerebellum. In conclusion, the results indicate that high dietary Pi can perturb normal brain growth, possibly through Akt-ERK signaling in developing mice.
Key Words: inorganic phosphate; brain growth; Akt; ERK; cap-dependent translation.
INTRODUCTION
Inorganic phosphate (Pi) is present in bacterial, fungal, plant, and animal cells. It plays a critical role in diverse cellular functions involving intermediary metabolism and energy-transfer mechanisms. It is a vital component of phospholipids in membrane and of nucleotides, both those which provide energy and those which serve as components of DNA, RNA, and phosphorylated intermediates in cellular signaling (Takeda et al., 2004). Regulation of Pi balance is accomplished by the family members of sodium-dependent inorganic phosphate transporter (NPT), which regulates entrance into the cellular membrane (Caverzasio and Bonjour, 1996). Neurons have been demonstrated to contain phosphate transporters in vitro (Glinn et al., 1995). Brain-specific NPT has been found to exist principally in cerebrum and cerebellum (Ni et al., 1996) and NPT2 in the third ventricle and amygdala (Mulroney et al., 2004). Though the biological significance of brain-specific NPT is still unknown, its role appears to be of paramount importance. Previous research has identified a wide range of nutritional and mineral risk factors that relate to cognitive function in children (Bryan et al., 2004; LaManna and Lust, 1997; Pollitt et al., 1996). Additionally, studies suggest that disturbances in ionic homeostasis may contribute to cerebral excitability (Uluitu et al., 1995) and secondary complications after brain injury (Reinert et al., 2000). An increased knowledge concerning the modified response of the brains of young animals to external stimuli and the effects of a disruption of homeostasis on normal cell function, survival, and growth may provide critical clues for coping with diverse changes, subtle or dramatic, in brain function.
The serine/threonine protein kinase Homo sapiens v-akt murine thymoma viral oncogene homolog 1 (Akt), also termed protein kinase B (PKB), controls key cellular processes such as glucose metabolism (Cross et al., 1995), cell cycle progression (Lawlor and Alessi, 2001), and apoptosis (Hovelmann et al., 2004). Akt has been identified as a key effector of the phosphatidylinositol-3-phosphate (PI3K) signaling pathway and functions to promote cell survival by inhibiting apoptosis through its ability to phosphorylate and inactivate several targets. Binding of PI3K-generated phospholipids to Akt results in translocation of Akt from the cytoplasm to the inner surface of the membrane, where Akt is phosphorylated (Kane et al., 2002). Several lines of recent evidence suggest that Akt is involved in various brain dysfunctions such as ischemia (Brywe et al., 2005), Alzheimer's disease (Onyango et al., 2005), and seizures (Cote et al., 2005), among other neurodegenerative diseases (Bauer et al., 2003).
Surveys conducted in various countries indicate that the intake of Pi has increased steadily as Pi-containing foods increased by approximately 17% in the decade leading up to 1993. These surveys also suggested that the use of Pi as a food additive may continue to increase (Calvo, 1993). To date, many studies involving Pi have focused mainly on its effect in bones and kidneys. However, studies have not yet investigated homeostatic maintenance of the brain and the brain's adaptation to excess Pi. The aim of this study, therefore, is to begin to elucidate the potential effects of high levels of Pi on the brains of developing mice. The results reveal that high dietary Pi perturbs the growth of the brain in young mice and alters Akt– extracellular regulated kinase (ERK) signaling. Furthermore, analysis of cap-dependent and internal ribosome entry site (IRES)-dependent translation using a CMV-renilla luciferase (LucR)-cMyc-IRES-firefly luciferase (LucF) construct suggests differences in protein translation in response to dietary phosphate. Our results support the hypothesis that Pi works as a stimulus capable of increasing or decreasing several pivotal genes for normal growth and suggest that regulation of Pi consumption is important in maintaining a high quality of life.
MATERIALS AND METHODS
Animals and diet.
Transgenic mice expressing CMV-LucR-c-myc-IRES-LucF reporter gene are convenient and powerful tools for the confirmation of cap-dependent and cap-independent protein translation since LucR and LucF provide the level of cap-dependent and cap-independent (IRES) protein translation, respectively (Creancier et al., 2001). A previous microarray and proteomic study suggested that high levels of phosphate may affect post-transcriptional protein translation (Conrads et al., 2005). In order to observe the effects of high levels of Pi on the brain growth of young mice 2-week-old transgenic male mice were used. The mice were separated into two dietary groups of five mice; one group received an AIN93-based diet containing 0.5% Pi (normal Pi), and the other group received the same diet fortified with 1.0% Pi (high Pi). All diets were prepared according to the guidelines of American Institute of Nutrition (AIN) and thus fulfill the requirement for normal growth, which is described in detail in Reeves et al. (1993). The mice were on the specified diet for 4 weeks until complete physical maturation (6 weeks after birth). At the end of the experiment, all mice were sacrificed, and blood was taken by cardiac puncture for further analysis. Also, the cerebellum and cerebrum were dissected under stereomicroscope and stored in liquid nitrogen for further use.
Serum biochemical and hematological analysis.
A blood chemistry test was carried out with the blood collected for serum biochemical and hematological analysis. The levels of calcium and phosphorus in the serum were determined using a biochemical autoanalyzer (VITALAB, Merck, The Netherlands). Hematological parameters consisting of erythrocyte, leukocyte, neutrophil, lymphocyte, monocyte, eosinophil, basophil, platelet, hemoglobin, and hematocrit were determined with a hematological autoanalyzer (Coulter T540 hematology system, Fullerton, CA, USA).
Luciferase assay.
Luciferase activities in the tissue extracts were measured by EG&G Berthold luminometer (Bundoora, Australia). Briefly, the dissected cerebellum and cerebrum were homogenized in passive lysis buffer (Promega, Madison, WI, USA). The homogenates were centrifuged for 20 min at 4500 rpm at 4°C and the supernatant was centrifuged for an additional 15 min at 13000 rpm at 4°C. LucF and LucR activities were measured using a dual luciferase assay kit (Promega).
Western blot analysis.
After measuring the protein concentration of the homogenized lysates using a Bradford kit (Bio-Rad, Hercules, CA, USA), equal amounts (50 μg) of protein were separated on sodium dodecyl sulfate–polyacrylamide gels (SDS-PAG) and transferred to nitrocellulose membranes. The membranes were blocked for 1 h in TBST (Tris–buffered saline + Tween 20) containing 5% skim milk; immunoblotting was performed by incubating the membranes overnight with their corresponding primary antibodies at 4°C. Antibodies raised against NPT1, p-Akt (Thr308, ser473), Akt1, p-ERK1/2, ERK1/2, 4E-BP1, proliferating cell nuclear antigen (PCNA), cyclin D1, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mammalian target of rapamycin (mTOR) was obtained from Cell Signaling (Beverly, MA, USA). After they were washed in TBST, the membranes were incubated with a horseradish peroxidase (HRP)-labeled secondary antibody, and the bands of interest were detected using a luminescent image analyzer, LAS-3000 (Fujifilm, Japan). Results were quantified using Multi Gauge version 2.02 program of the LAS-3000.
Immunoprecipitation and kinase assay.
A decrease in phosphorylation of mTOR is often associated with decreased kinase activity. To determine if this was the case in response to high Pi, mTOR from mouse brain lysate was immunoprecipitated and kinase activity assay was performed with 4E-BP1 phosphorylated heat- and acid-stable (PHAS-1) protein as a substrate. Immunoprecipitation of mTOR was carried out using the Seize Primary Mammalian Immunoprecipitation Kit according to manufacturer's guidelines (Pierce, Rockford, IL, USA). The mTOR kinase assay was performed with 300 μM ATP and 1 μg PHAS-1 (Calbiochem, San Diego, CA) for 30 min at 30°C. Reactions were terminated by adding 5x sample buffer and boiling. The samples were analyzed by 15% SDS/PAG electrophoresis.
Immunohistochemistry.
Formalin-fixed, paraffin-embedded tissue sections (5 μm) were transferred to plus slides (Fisher Scientific, Pittsburgh, PA, USA). The tissue sections were deparaffinized in xylene and rehydrated through alcohol gradients and incubated in 200 μl of proteinase K, then washed and incubated in 0.3% hydrogen peroxide (AppliChem, Darmstadt, Germany) for 30 min to quench endogenous peroxidase activity. After they were in PBS, the tissue sections were incubated with 5% BSA in PBS for 1 h at room temperature to block unspecific binding sites. Primary antibodies were applied on tissue sections overnight at 4°C. The following day, the tissue sections were washed and incubated with secondary HRP-conjugated antibodies for 1 h at room temperature. After careful washing, tissue sections were counterstained with Mayer's hematoxylin (DAKO, Caepinteria, CA, USA) and washed with xylene. Cover slips were mounted using Permount (Fisher, Pittsburgh, PA, USA), and the slides were reviewed in a light microscope (Carl Zeiss, Thornwood, NY, USA).
Data analysis.
Quantification of Western blot analysis was performed using Multi Gauge version 2.02 program (Fujifilm). All results are given as means ± SE. Result were analyzed by Student's t-test (Graphpad Software, San Diego, CA, USA) or two–way analysis of variance (ANOVA; SAS User Guide Statistics, SAS Institute, Cary, NC, USA). A value of p < 0.05 was considered significant (), and p < 0.01 was highly significant () compared to the corresponding control.
RESULTS
High Dietary Levels of Pi Perturb Growth of Cerebellum of Young Mice
The diverse functional roles of Pi and different nutritional requirements during growth suggest that appropriate in vivo nutritional studies at specific stages of growth are needed. Therefore, the potential effects of high levels of Pi on the development of young mice were investigated. A high Pi diet caused reduced body weight after 3 weeks of treatment, although food intake was equivalent to normal food consumption and growth rate was similar (Fig. 1A). A High Pi diet decreased the relative weight of cerebrum slightly; however, the relative weight of cerebellum was significantly decreased (Fig. 1B). A high Pi diet significantly increased the serum phosphate level and significantly decreased calcium concentration as compared to that of the normal diet group (Fig. 1C). Other serum biochemical and hematological values remained within normal range (data not shown).
A High Level of Dietary Pi Increases Brain-Specific NPT1
To confirm the hypothesis that phosphate transport in the brain may play a key role in the homeostasis of brain function as mentioned earlier, the potential effect of high dietary Pi on brain-specific NPT protein expression was evaluated. A high Pi diet significantly increased NPT1 protein expression in both cerebrum and cerebellum (Fig. 2A, B). Densitometric analysis clearly demonstrated how high levels of dietary and serum Pi increased NPT1 protein expression in the test group (Fig. 2C, D).
High Levels of Dietary Pi Regulate Akt Phosphorylation and Akt-Related Signals
Akt is involved in a wide range of important functions in diverse organs, including the brain, and it has been demonstrated to be an important signaling protein in response to nutrient metabolism (Song et al., 2005 and references therein). This fact and the results of our preliminary study, which indicated that high levels of Pi have the potential to change the pattern of Akt protein expression in mouse brains, prompted us to investigate the effects of high levels of Pi on Akt-related signals in vivo. High levels of dietary Pi significantly increased Akt phosphorylation at Ser473 in cerebellum; however, no significant changes were observed in cerebrum. In contrast, high levels of Pi decreased Akt phosphorylation at Thr308 in both cerebrum and cerebellum (Fig. 3A, B). Densitometric analysis (Fig. 3C, D) indicates that for total Akt expression, high dietary Pi decreased protein expression in cerebrum, while that of cerebellum remained unchanged (Fig. 3A, B). In this study, phosphospecific antibodies were employed to measure Akt activation; Akt is activated by phosphorylation (reviewed in Song et al., 2005). To control for loading or changes in total Akt protein the level of Akt activation is represented as a ratio of phosphorylated to total protein. As shown in Figure 3E and F, high levels of Pi increased Akt phosphorylation at Ser473 in both cerebrum and cerebellum but did not affect Akt phosphorylation at Thr308 in cerebrum, although a significant suppression of Akt activity was observed in cerebellum. Such data suggest that high Pi regulates Akt phosphorylation in cerebrum and cerebellum in a somewhat different manner. Our results were further confirmed by immunohistochemical analysis. High levels of Pi decreased the expression of p-Akt (Thr308) compared to that of the normal diet group, especially in cerebellum (Fig. 4A, B). However, high levels of Pi increased the expression of p-Akt (Ser473) in both cerebrum and cerebellum (Fig. 4A, B). To investigate whether high Pi affects Akt-related signal pathways, changes in the appropriate Akt-related signals were measured. High levels of Pi slightly reduced protein expression of mTOR in both regions of the brain, even though there was no statistical significance. In contrast, a change in the expression pattern of the eIF4E-BP1 protein was evident. High levels of Pi significantly decreased eIF4E-BP1 protein expression in cerebrum but did not induce any change in cerebellum (Fig. 5A, B, C, D).
High Dietary Pi Regulate Protein Translation
The data above showing Pi-mediated selective suppression of eIF4E-BP1 in cerebrum strongly suggest that high levels of Pi may regulate cap-dependent protein translation. In fact, recent lines of evidence suggest that cap-dependent protein translation regulation has immense biological significance, and affects such processes as cell growth and development, oncogenic transformation and even the function of the central nervous system (Richter and Sonenberg, 2005). Therefore, the effects of high levels of Pi on protein translation in young mouse brains were closely examined. High levels of Pi slightly increased mTOR kinase activity in cerebrum, but the activity remained unchanged in cerebellum (Fig. 6A, B). A minimal or no change in mTOR kinase activity was observed in cerebrum and cerebellum in response to high levels of Pi, but an increase in cap-dependent protein translation was visible in cerebrum. Firefly luciferase and renilla luciferase assays were performed to determine the levels of cap-independent and cap-dependent protein translation. Results indicated that high levels of Pi enhanced cap-dependent protein translation in cerebrum, more so than in cerebellum, where IRES-mediated protein translation was dominant (Fig. 6C, D).
High Dietary Pi Affects ERK Phosphorylation but Not Mnk Activities
Because ERK is known to negatively regulate eIF4E-BP1 protein expression through Mnk1 (Rolli-Derkinderen et al., 2003; Rosenwald, 2004), and because Akt is closely associated with the ERK pathway (Perkinton et al., 2002), the potential effects of high levels of Pi on ERK and Mnk signals were evaluated. Phosphospecific antibodies were employed to measure the activity of ERK, and suppression of ERK phosphorylation in response to high levels of Pi was observed both in cerebrum and cerebellum. Interestingly, high levels of Pi reduced phosphorylation of ERK1 (p44)—more so than that of ERK2 (p42)—but no significant change was observed in the total ERK1/2 (Fig. 7A, B). High levels of Pi caused a significant reduction in ERK1 phosphorylation over total ERK1 and a slight reduction in ERK2 phosphorylation over total ERK2 in cerebrum (Fig. 7C). A similar pattern was observed in cerebellum. Only ERK1 phosphorylation over total ERK1 was decreased significantly in cerebellum (Fig. 7D). In contrast, Western blot analysis revealed that high levels of Pi did not induce any change in Mnk activity (data not shown).
High Levels of Pi Decreased Cell Proliferation through Suppression of Cyclin D1 and PCNA
Inorganic phosphorous is known as a signaling molecule capable of regulating global gene expression and cellular functioning in multiple cell types (Beck et al., 2003). Therefore, this study was undertaken to evaluate the effects of high levels of Pi on brain cell proliferation, which was investigated by Western blot analysis, measuring the expression of marker proteins characteristic of cell cycle such as PCNA and cyclin D1. The study found that high levels of Pi caused a significant suppression of the protein expression of both PCNA and cyclin D1 in both regions of the brain (Fig. 8A, B, C, D).
DISCUSSION
Serum Pi level is maintained within a narrow range through complex interaction between intestinal absorption, exchange with intracellular and bone storage pools, and renal tubular reabsorption. As mentioned earlier, Pi plays a key role in diverse physiological functions. Recently, the NPT specific to the brain has been identified, and considerable progress has been made in our understanding of their function and regulation (Hayashi et al., 2001; Hisano, 2002). Pi transport into the brain is regulated mainly by dietary and serum Pi (Takeda et al., 2004). Research indicates that Pi works as a stimulus capable of increasing or decreasing the expression of a number of pivotal genes, such as those involved in the regulation of transcription, signal transduction, and cell cycle (Beck, 2003). It is thought that an excess of Pi intake over a long period of time is a strong factor in aging (Takeda et al., 2004). Therefore, restricting Pi consumption seems to be important in maintaining good quality of life. These reasons, the potential importance of Pi as a novel signaling molecule, and the risk of diverse neurodegenerative diseases that involve brain-specific NPT1, prompted us to define the pathways by which Pi affects the growth of young mouse brain.
In this study, the correlation between growth and high levels of Pi exposure during the development of weanling mice was examined. The high Pi diet group demonstrated reduced body weight (Fig. 1A) in addition to decreased relative brain weight (Fig. 1B), suggesting the importance of phosphate levels in specific tissues and in overall normal growth. The AIN93 diet composition itself used in this study should not affect the animals' growth, because its composition fulfills the requirements for normal growth in laboratory animals; thus we can exclude the possibility of malnutrition (Reeves et al., 1993) and consider the possibility that factors other than malnutrition could be critical for causing the growth retardation. For example, high serum phosphate levels could contribute to the development of arteriosclerosis and could lead to secondary hyperparathyroidism, both of which are detrimental to health and hinder normal development (Sitara et al., 2004). Significant reductions in serum Ca2+ levels in response to high levels of Pi may also shed light on the detrimental effects of high levels of Pi on normal growth (Fig. 1C). In fact, calcium is responsible for the tight regulation of the secretion of parathyroid hormone (PTH), an excess of which results in hyperparathyroidism. To maintain an appropriate level of PTH, its degradation normally occurs within the parathyroid gland prior to secretion (Shoback et al., 2003). In this study, we hypothesize that high Pi–induced reduction of serum Ca2+ interferes with normal degradation of PTH, and in turn retards growth. This hypothesis is supported by a recent study that demonstrated how low concentrations of extracellular Ca2+ downregulate degradation of PTH through the suppression of (1–84)hPTH generation, which is responsible for N-terminal truncation of PTH in parathyroid cells (Kawata et al., 2005). It can therefore be hypothesized that high Pi–induced reductions of serum Ca2+ levels may lead to hyperparathyroidism, which interferes with normal growth through bone resorption by impairing degradation of PTH.
Akt is a potent molecule that has been shown to be necessary for the survival of a variety of neurons and non-neuronal cells. A recent study has also demonstrated that adhesion molecules on glia play an important role in the regulation of brain growth through Akt phosphorylation (Scheidenhelm et al., 2005). Both of these facts suggest that a disruption in Akt activity may have an adverse effect on normal development of the brain. Akt is activated by phosphorylation at two important sites, Thr308 and Ser473 (Kandel and Hay, 1999). Phosphorylation at both sites is required for full activity, and suppression of phosphorylation at either site can induce abrogation of tumor development (Kim et al., 2004). Results of our study show an increase in high Pi-induced Akt phosphorylation at Ser473 in both cerebrum and cerebellum, but a decrease in phosphorylation at Thr308 in cerebellum (Fig. 3E, F). In addition, other data show that reduced activity of Akt due to an altered pattern of phosphorylation is coupled with a significant decrease in protein expressions of PCNA and cyclin D1 (Fig. 8). These results strongly suggest that high levels of Pi may affect brain growth by disturbing Akt activity through different modes of action in cerebrum and cerebellum. Recent evidence indicates that the Akt family has three highly conserved proteins as members, namely, Akt1, Akt2, and Akt3, the first two of which are required for normal growth and metabolism, respectively (Easton et al., 2005). Akt1, required for normal growth, was used in this study, and decreased Akt activity in response to high levels of Pi seemed to disturb the proliferation of brain cells somewhat differently in cerebrum and cerebellum. This correlation has also been noted in research conducted by Easton et al., where Akt1- and Akt3-deficient brains were reduced in size by approximately the same amount. Furthermore, the absence of Akt1 led to a reduction in cell numbers, whereas the lack of Akt3 resulted in smaller and fewer cells. Additionally, attenuated mTOR signaling was observed in the brains of Akt3-deficient mice but not in Akt1-deficient mouse brains (Easton et al., 2005).
Our results also indicate that high levels of dietary Pi do not have a significant effect on the levels of mTOR in either region of the mouse brain were Akt phosphorylation is downregulated (Fig. 5). Therefore, high levels of Pi may affect cell proliferation, not through Akt1, but through other types of Akt. In effect, high levels of Pi may downregulate brain cell growth through disturbed Akt activity; however, the mode of action in cerebrum may be somewhat different from that of cerebellum. Therefore, the mechanisms by which high Pi induces the disruption of Akt activity in cerebrum and cerebellum should be further elucidated, and extensive efforts to reveal such mechanisms are under way.
As discussed previously, Akt signaling is closely associated with the ERK pathway (Rosenwald, 2004). Tests were performed in our study to examine whether high levels of Pi affect ERK and Mnk1 in the brain. Results indicate that in response to high dietary levels of Pi, ERK1 activation is significantly downregulated in both cerebrum and cerebellum, whereas no significant change in ERK2 activation was observed (Fig. 7C, D). It has been demonstrated that transgenic mice overexpressing phosphorylated ERK1/2 in the brain are protected against oxygen neurotoxicity (Felderhoff-Mueser et al., 2004). Such activated ERK may be partially responsible for the protection of brain cells against oxidative stress. In our study, high dietary Pi suppressed ERK activation, especially ERK1. This suggests that long-term exposure to excess dietary Pi may increase the susceptibility of the young mouse brain to xenobiotic stimuli.
The regulation of cyclin D1 expression can occur at many levels (Perkinton et al., 2002), but whether expression of cyclin D1 is regulated at a step that can be strictly defined as a translation stage, has not been determined. Expression of cyclin D1 can be induced independently either through the PI3K (Takuwa et al., 1999) or the ERK (Lavoie et al., 1996) pathway. However, a recent study has revealed crosstalk between Akt and the ERK signals in cultured rat cerebellar granule cells (Myhre et al., 2004), suggesting that the suppressed expression of cyclin D1 in cerebrum and cerebellum in our study could also result from crosstalk between Akt and ERK signals. In fact, reduced phosphorylation of Thr308 and Ser473 of Akt (Fig. 3E, F), and the selective suppression of ERK1 phosphorylation (Fig. 7C, D) that occurred simultaneously, may represent a mechanism to explain the impairment of brain cell growth in our study. Put succinctly, high levels of Pi may increase the susceptibility of brain cells to external stimuli through crosstalk between Akt and ERK, and reduced serum Ca2+ levels may aggravate the process.
It was observed in the present study that high Pi–stimulated cap-dependent translation occurs in cerebrum, whereas cap-independent translation takes place in cerebellum (Fig. 6C, D). Selective augmentation of Akt phosphorylation at Ser473 in cerebrum (Fig. 3E) and an increase in Akt phosphorylation at Ser473 coupled with a decrease in Akt phosphorylation at Thr308 in cerebellum (Fig. 3F), may be partially responsible for the differential translation. In fact, differential regulation of cap-independent translation and decreased cyclin D1 expression may account for the differential sensitivity of high-vs-low Akt activity (Shi et al., 2005).
Current research suggests that cyclin D1 is normally accumulated during the G2 phase of the cell cycle and that synthesis during this phase may factor into the rapid rise in cyclin D1 to the level required to initiate G1 transit in actively proliferating cells (Sherr, 2002). The reduction in cap-dependent protein synthesis during the G2/M cell cycle transition has also been recently underscored (Pyronnet et al., 2001). Our data imply that Akt-related cyclin D1 reduction is closely associated with ERK inactivation. Experiments designed to elaborate on this implication are under way. Our results support a working model in which Akt-dependent control of cyclin D1 can regulate the expression of critical determinants for G1 arrest or aging.
In summary, the present study suggests that high levels of dietary Pi may have detrimental effects on the normal growth of young mouse brains through the disruption of Akt-ERK signals. The Akt-ERK signaling pathway may also be involved in numerous biological processes critical to normal development and may modulate various neurodegenerative diseases of the brain. Additionally, elevated dietary Pi caused a shift in cap-dependent verse cap-independent translation, which also may negatively affect tissue function. Extensive studies to elucidate the precise effects of such activated signals on both development and pathogenesis in the brain are in progress.
ACKNOWLEDGMENTS
This work was supported by grants from the Korea Science and Engineering Foundation (KOSEF). J.H., S.K.H., and K.Y. are also grateful for the award of the BK21 fellowship. K.H.L. was supported by the grants from 21C Frontier Functional Human Genome Project and National Nuclear R&D Program from Ministry of Science and Technology GRB. Jr. was supported by a National Cancer Institute Grant (CA084573).
REFERENCES
Bauer, M., Alda, M., Priller, J., and Young, L. T. The International Group for the Study of Lithium Treated Patients (IGSLI). (2003). Implications of the neuroprotective effects of lithium for the treatment of bipolar and neurodegenerative disorders. Pharmacopsychiatry 36(Suppl. 3), S250–S254.
Beck, G. R., Jr. (2003). Inorganic phosphate as a signaling molecule in osteoblast differentiation. J. Cell. Biochem. 90, 234–243.
Beck, G. R., Moran, E., and Knecht, N. (2003). Inorganic phosphate regulates multiple genes during osteoblast differentiation, including Nrf2. Exp.Cell Res. 288, 288–300.
Bryan, J., Osendarp, S., Hughes, D., Calvaresi, E., Baghurst, K., and van Klinken, J. W. (2004). Nutrients for cognitive development in school-aged children. Nutr. Rev. 62, 295–306.
Brywe, K. G., Mallard, C., Gustavsson, M., Hedtjarn, M., Leverin, A. L., Wang, X., Blomgren, K., Isgaard, J., and Hagberg, H. (2005). IGF-I neuroprotection in the immature brain after hypoxia–ischemia, involvement of Akt and GSK2beta Eur. J. Neurosci. 21, 1489–1502.
Calvo, M. S. (1993). Dietary phosphorous, calcium metabolism, and bone. J. Nutr. 123, F1627–F1633.
Caverzasio, J., and Bonjour, J. P. (1996). Characteristics and regulation of Pi transport in estrogenic cells for bone metabolism. Kidney Int. 49, 975–980.
Conrads, K. A., Yi, M., Simpson, K. A., Lucas, D. A., Camalier, C. E., Yu, L. R., Veenstra, T. D., Stephens, R. M., Conrads, T. P., and Beck, G. R., Jr. (2005). A combined proteome and microarray investigation of inorganic phosphate-induced pre-osteoblast cells. Mol. Cell Proteomics 9, 1284–1296.
Cote, A., Chiasson, M., Perelta, M. R., 3rd, Lafortune, K., Pellegrini, L., and Toth, K. (2005). Cell type–specific action of seizure-induced intracellular zinc communication in the rat hippocampus. J. Physiol. 566, 821–837.
Creancier, L., Mercier, P., Prats, A., and Morello, D. (2001). C-myc internal ribosome entry site activity is developmentally controlled and subjected to a strong translational repression in adult transgenic mice. Mol. Cell. Biol. 21, 1833–1840.
Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., and Hemmings, B. A. (1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378, 785–789.
Easton, R. M., Cho, H., Roovers, K., Shineman, D. W., Mizrahi, M., Forman, M. S., Lee, V. M., Szabolcs, M., de Jong, R., Oltersdorf, T., et al. (2005). Role of Akt3/protein kinase B gamma in attainment of normal brain size. Mol. Cell. Biol. 25, 1869–1878.
Felderhoff-Mueser, U., Bittigau, P., Sifringer, M., Jarosz, B., Korowicz, E., Mahler, L., Piening, T., Moysich, A., Grune, T., Thor, F., et al. (2004). Oxygen causes cell death in the developing brain. Neurobiol. Dis. 17, 273–282.
Glinn, M., Ni, B., and Paul, S. M. (1995). Characterization of Na(+)-dependent phosphate uptake in cultured fetal rat cortical neurons. J. Neurochem. 65, 2358–2365.
Hayashi, M., Otsuka, M., Morimoto, R., Hirota, S., Yatsushiro, S., Takeda, J., Yamamoto, A., and Moriyama, Y. (2001). Differentiation-associated Na+-dependent inorganic phosphate cotransporter (DNPI) is a vesicular glutamate transporter in endocrine glutamatergic systems. J. Biol. Chem. 276, 43400–43406.
Hisano, S. (2002). The inorganic phosphate transport system in the brain and the potency as vesicular glutamate transporters. Kaibogaku Zasshi 77, 51–58.
Hovelmann, S., Beckers, T. L., and Schmidt, M. (2004). Molecular alterations in apoptotic pathways after PKB/Akt-mediated chemoresistance in NCI H460 cells. Br. J. Cancer 90, 2370–2377.
Kandel, E. S., and Hay, N. (1999). The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB. Exp. Cell Res. 253, 210–229.
Kane, L. P., Mollenauer, M. N., Xu, Z., Turck, C. W., and Weiss, A. (2002). Akt-dependent phosphorylation specifically regulates Cot induction of NF-kappa B-dependent transcription. Mol. Cell. Biol. 22, 5962–5974.
Kawata, T., Imanishi, Y., Konayashi, K., Onoda, N., Takemoto, Y., Tahara, H., Okuno, S., Ishimura, E., Miki, T., Ishikawa, T., et al. (2005). Direct in vitro evidence of extracellular Ca2+ induced N-terminal truncation of (1–84)hPTH by human parathyroid cells. J. Clin. Endocrinol. Metab. 90, 5774–5778.
Kim, H. W., Park, I. K., Cho, C. S., Lee, K. H., Beck, G. R., Colburn, N. H., and Cho, M. H. (2004). Aerosol gene delivery of glucosylated polyethyleneimine (GPEI)/PTEN complex suppresses Akt downstream pathways in the lung of K-ras null mice. Cancer Res. 64, 7971–7976.
LaManna, J. C., and Lust, W. D. (1997). Nutrient consumption and metabolic perturbations. Neurosurg Clin North Am. 8, 145–163.
Lavoie, J. N., L'Allemain, G., Brunet, A., Muller, R., and Pouyssegur, J. (1996). Cyclin D1 expression is regulated positively by the p42/p44 MAPK and negatively by the p38/HOGMAPK pathway. J. Biol. Chem. 271, 20608–20616.
Lawlor, M. A., and Alessi, D. R. (2001). PKB/Akt: A key mediator of cell proliferation, survival and insulin responses. J. Cell Sci. 114, 2903–2910.
Mulroney, S. E., Woda, C. B., Halaihel, N., Louie, B., McDonnell, K., Schulkin, J., Haramati, A., and Levi, M. (2004). Central control of renal sodium-phosphate (NaPi-2) transporters. Am. J. Physiol. Renal Cell Physiol. 286, F647–F652.
Myhre, O., Sterri, S. H., Bogen, I. L., and Fonnum, F. (2004). ERK1/2 phosphorylation and reactive oxygen species formation via nitric oxide and Akt-1/Raf-1 crosstalk in cultured cerebellar granule cells exposed to the organic solvent 1,2,4-trimethylcyclohexane. Toxicol.Sci. 80, 296–303.
Ni, B., Du, Y., Wu, X., DeHoff, B. S., Rostek, P. R., and Paul, S. M. (1996). Molecular cloning, expression, and chromosomal localization of a human brain-specific Na+-dependent inorganic phosphate cotransporter. J. Neurochem. 66, 2227–2238.
Onyango, I. G., Bennett, J. P., Jr., and Tuttle, J. B. (2005). Endogenous oxidative stress in sporatic Alzheimer/s disease neuronal cybrids reduces viability by increasing apoptosis through pro-death signaling pathways and is mimicked by oxidant exposure of control cybrids. Neurobiol. Dis. 19, 312–322.
Perkinton, M. S., Ip, J. K., Wood, G. L., Crossthwaite, A. J., and Williams, R. J. (2002). Phosphatidylinositol 3-kinase is a central mediator of NMDA receptor signaling to MAP kinase (ERK1/2), Akt/PKB and CREB in striatal neurons. J. Neurochem. 80, 239–254.
Pollitt, E., Jacoby, E., and Cueto, S. (1996). School breakfast and cognition among nutritionally at-risk children in the Peruvian Andes. Nutr. Rev. 54, S22–S26.
Pyronnet, S., Dosite, J., and Sonnenberg, N. (2001). Suppression of cap-dependent translation in mitosis. Genes Dev. 15, 2083–2093.
Reeves, P. G., Nielsen, F. H., and Fahey, G. C., Jr. (1993). AIN-93 purified diets for laboratory rodents: Final report of the American Institute of Nutrition ad hoc Writing Committee on the Reformulation of the AIN-76A rodent diet. J. Nutr. 123, 1939–1951.
Reinert, M., Khaldi, A., Zauner, A., Doppenberg, E., Choi, S., and Bullock, R. (2000). High level of extracellular potassium and its correlates after severe head injury: Relationship to high intracranial pressure. J. Neurosurg. 93, 800–807.
Richter, J. D., and Sonenberg, N. (2005). Regulation of cap-dependent translation by eIF4E inhibitory proteins. Nature 433, 477–480.
Rolli-Derkinderen, M., Machavoine, F., Baraban, J. M., Grolleau, A., Beretta, L., and Dy, M. (2003). ERK and p38 inhibit the expression of 4E-BP1 repressor of translation through induction of Egr-1. J. Biol. Chem. 278, 18859–18867.
Rosenwald, I. B. (2004). The role of translation in neoplastic transformation from a pathologist's point of view. Oncogene 23, 3230–3237.
Scheidenhelm, D. K., Cresswell, J., Haipek, C. A., Fleming, T. P., Mercer, R. W., and Gutmann, D. H. (2005). Akt-dependent cell size regulation by the adhesion molecule on glia occurs independently of phosphatidylinositol-3-kinase and Rheb signaling. Mol. Cell. Biol. 25, 3151–3162.
Sherr, C. J. (2002). D1 in G2. Cell Cycle 1, 36–38.
Shi, Y., Sharma, A., Wu, H., Lichtenstein, A., and Gera, J. (2005). Cyclin D1 and c-myc internal ribosome entry site (IRES)-dependent translation is regulated by Akt activity and enhanced by rapamycin through a p38 MAPK and ERK-dependent pathway. J. Biol. Chem. 280, 10964–10973.
Shoback, D. M., Bilezikian, J. P., Turner, S. A., McCary, L. C., Guo, M. D., and Peacock, M. (2003). The calcimimetic cinacalcet normalizes serum calcium in subjects with primary hyperthyroidism. J. Clin. Endocrinol. Metab. 88, 5644–5649.
Sitara, D., Razzaque, M. S., Hesse, M., Yoganathan, S., Taguchi, T., Erben, R. G., Juppner, H., and Lanske, B. (2004). Homozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverses hypophosphatemia in Phex-deficient mice. Matrix Biol. 23, 421–432.
Song, G., Ouyang, G., and Bao, S. (2005). The activation of Akt/PKB signaling pathway and cell survival. J. Cell Mol. Med. 9, 59–71.
Takeda, E., Yamamoto, H., Nashiki, K., Sato, T., Arai, H., and Taketani, Y. (2004). Inorganic phosphate homeostasis and the role of dietary phosphorus. J. Cell. Mol. Med. 8, 191–200.
Takuwa, N., Fukui, Y., and Takuwa, Y. (1999). Cyclin D1 expression mediated by phosphatidylinositol-3-kinase through mTOR-p70S6K-independent signaling in growth factor-stimulated NIH3T3 fibroblasts. Mol. Cell. Biol. 19, 1346–1358.
Uluitu, M., Badescu, A., and Chis, R. (1995). Influence of increased sodium intake on cerebral excitability. Rom. J. Physiol. 32, 11–33.(Hua Jin, Soon-Kyung Hwang, Kyungnam Yu, )
Department of Food and Nutrition, College of Human Ecology, Seoul National University, Seoul 151–742, Korea
Laboratory of Molecular Oncology, Korea Institute of Radiological & Medical Sciences, Seoul 139–240, Korea
Institut National de la Sante et de la Recherche Medicale U589, Institut Louis Bugnard, IFR31, CHU Rangueil, Batiment L3, Avenue Jean Poulhes, BP 84225, 31432 Toulouse Cedex 4, France
Centre de Biologie du Developpement, CNRS-UMR5547, IFR 109, Universite Paul Sabatier, 31062 Toulouse, France
Emory University School of Medicine, Division of Endocrinology, Metabolism and Lipids, Atlanta GA 30322, USA
ABSTRACT
Inorganic phosphate (Pi) plays a key role in diverse physiological functions. Recently, considerable progress has been made in our understanding of the function and regulation of the brain-specific sodium-dependent inorganic phosphate transporter 1 (NPT1), which is found to exist principally in cerebrum and cerebellum. The potential importance of Pi as a novel signaling molecule and the poor prognosis of diverse neurodegenerative diseases that involve brain-specific NPT1 have prompted us to define the pathways by which Pi affects mouse brain growth. A high phosphate diet caused an increase in serum Pi accompanied by a decrease in calcium, and a decrease in body weight coupled with a decreased relative weight of cerebellum. A high phosphate diet caused a significant increase in protein expression of NPT1, both in cerebrum and cerebellum. Additionally, the high phosphate diet increased Homo sapiens v-akt murine thymoma viral oncogene homolog 1 (Akt) phosphorylation at Ser473 in cerebrum and cerebellum, whereas suppression of Akt phosphorylation at Thr308 was observed only in cerebellum. Selective suppression of eukaryotic translation initiation factor-binding protein (eIF4E-BP1) in cerebrum was induced by high levels of Pi, which induced cap-dependent and cap-independent protein translation in cerebrum and cerebellum, respectively. Phosphorylation of extracellular regulated kinase 1 (ERK1) in comparison with that of ERK2 was significantly reduced in both cerebrum and cerebellum. High levels of Pi reduced protein expressions of proliferating cell nuclear antigen (PCNA) and cyclin D1 in cerebrum and cerebellum. In conclusion, the results indicate that high dietary Pi can perturb normal brain growth, possibly through Akt-ERK signaling in developing mice.
Key Words: inorganic phosphate; brain growth; Akt; ERK; cap-dependent translation.
INTRODUCTION
Inorganic phosphate (Pi) is present in bacterial, fungal, plant, and animal cells. It plays a critical role in diverse cellular functions involving intermediary metabolism and energy-transfer mechanisms. It is a vital component of phospholipids in membrane and of nucleotides, both those which provide energy and those which serve as components of DNA, RNA, and phosphorylated intermediates in cellular signaling (Takeda et al., 2004). Regulation of Pi balance is accomplished by the family members of sodium-dependent inorganic phosphate transporter (NPT), which regulates entrance into the cellular membrane (Caverzasio and Bonjour, 1996). Neurons have been demonstrated to contain phosphate transporters in vitro (Glinn et al., 1995). Brain-specific NPT has been found to exist principally in cerebrum and cerebellum (Ni et al., 1996) and NPT2 in the third ventricle and amygdala (Mulroney et al., 2004). Though the biological significance of brain-specific NPT is still unknown, its role appears to be of paramount importance. Previous research has identified a wide range of nutritional and mineral risk factors that relate to cognitive function in children (Bryan et al., 2004; LaManna and Lust, 1997; Pollitt et al., 1996). Additionally, studies suggest that disturbances in ionic homeostasis may contribute to cerebral excitability (Uluitu et al., 1995) and secondary complications after brain injury (Reinert et al., 2000). An increased knowledge concerning the modified response of the brains of young animals to external stimuli and the effects of a disruption of homeostasis on normal cell function, survival, and growth may provide critical clues for coping with diverse changes, subtle or dramatic, in brain function.
The serine/threonine protein kinase Homo sapiens v-akt murine thymoma viral oncogene homolog 1 (Akt), also termed protein kinase B (PKB), controls key cellular processes such as glucose metabolism (Cross et al., 1995), cell cycle progression (Lawlor and Alessi, 2001), and apoptosis (Hovelmann et al., 2004). Akt has been identified as a key effector of the phosphatidylinositol-3-phosphate (PI3K) signaling pathway and functions to promote cell survival by inhibiting apoptosis through its ability to phosphorylate and inactivate several targets. Binding of PI3K-generated phospholipids to Akt results in translocation of Akt from the cytoplasm to the inner surface of the membrane, where Akt is phosphorylated (Kane et al., 2002). Several lines of recent evidence suggest that Akt is involved in various brain dysfunctions such as ischemia (Brywe et al., 2005), Alzheimer's disease (Onyango et al., 2005), and seizures (Cote et al., 2005), among other neurodegenerative diseases (Bauer et al., 2003).
Surveys conducted in various countries indicate that the intake of Pi has increased steadily as Pi-containing foods increased by approximately 17% in the decade leading up to 1993. These surveys also suggested that the use of Pi as a food additive may continue to increase (Calvo, 1993). To date, many studies involving Pi have focused mainly on its effect in bones and kidneys. However, studies have not yet investigated homeostatic maintenance of the brain and the brain's adaptation to excess Pi. The aim of this study, therefore, is to begin to elucidate the potential effects of high levels of Pi on the brains of developing mice. The results reveal that high dietary Pi perturbs the growth of the brain in young mice and alters Akt– extracellular regulated kinase (ERK) signaling. Furthermore, analysis of cap-dependent and internal ribosome entry site (IRES)-dependent translation using a CMV-renilla luciferase (LucR)-cMyc-IRES-firefly luciferase (LucF) construct suggests differences in protein translation in response to dietary phosphate. Our results support the hypothesis that Pi works as a stimulus capable of increasing or decreasing several pivotal genes for normal growth and suggest that regulation of Pi consumption is important in maintaining a high quality of life.
MATERIALS AND METHODS
Animals and diet.
Transgenic mice expressing CMV-LucR-c-myc-IRES-LucF reporter gene are convenient and powerful tools for the confirmation of cap-dependent and cap-independent protein translation since LucR and LucF provide the level of cap-dependent and cap-independent (IRES) protein translation, respectively (Creancier et al., 2001). A previous microarray and proteomic study suggested that high levels of phosphate may affect post-transcriptional protein translation (Conrads et al., 2005). In order to observe the effects of high levels of Pi on the brain growth of young mice 2-week-old transgenic male mice were used. The mice were separated into two dietary groups of five mice; one group received an AIN93-based diet containing 0.5% Pi (normal Pi), and the other group received the same diet fortified with 1.0% Pi (high Pi). All diets were prepared according to the guidelines of American Institute of Nutrition (AIN) and thus fulfill the requirement for normal growth, which is described in detail in Reeves et al. (1993). The mice were on the specified diet for 4 weeks until complete physical maturation (6 weeks after birth). At the end of the experiment, all mice were sacrificed, and blood was taken by cardiac puncture for further analysis. Also, the cerebellum and cerebrum were dissected under stereomicroscope and stored in liquid nitrogen for further use.
Serum biochemical and hematological analysis.
A blood chemistry test was carried out with the blood collected for serum biochemical and hematological analysis. The levels of calcium and phosphorus in the serum were determined using a biochemical autoanalyzer (VITALAB, Merck, The Netherlands). Hematological parameters consisting of erythrocyte, leukocyte, neutrophil, lymphocyte, monocyte, eosinophil, basophil, platelet, hemoglobin, and hematocrit were determined with a hematological autoanalyzer (Coulter T540 hematology system, Fullerton, CA, USA).
Luciferase assay.
Luciferase activities in the tissue extracts were measured by EG&G Berthold luminometer (Bundoora, Australia). Briefly, the dissected cerebellum and cerebrum were homogenized in passive lysis buffer (Promega, Madison, WI, USA). The homogenates were centrifuged for 20 min at 4500 rpm at 4°C and the supernatant was centrifuged for an additional 15 min at 13000 rpm at 4°C. LucF and LucR activities were measured using a dual luciferase assay kit (Promega).
Western blot analysis.
After measuring the protein concentration of the homogenized lysates using a Bradford kit (Bio-Rad, Hercules, CA, USA), equal amounts (50 μg) of protein were separated on sodium dodecyl sulfate–polyacrylamide gels (SDS-PAG) and transferred to nitrocellulose membranes. The membranes were blocked for 1 h in TBST (Tris–buffered saline + Tween 20) containing 5% skim milk; immunoblotting was performed by incubating the membranes overnight with their corresponding primary antibodies at 4°C. Antibodies raised against NPT1, p-Akt (Thr308, ser473), Akt1, p-ERK1/2, ERK1/2, 4E-BP1, proliferating cell nuclear antigen (PCNA), cyclin D1, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mammalian target of rapamycin (mTOR) was obtained from Cell Signaling (Beverly, MA, USA). After they were washed in TBST, the membranes were incubated with a horseradish peroxidase (HRP)-labeled secondary antibody, and the bands of interest were detected using a luminescent image analyzer, LAS-3000 (Fujifilm, Japan). Results were quantified using Multi Gauge version 2.02 program of the LAS-3000.
Immunoprecipitation and kinase assay.
A decrease in phosphorylation of mTOR is often associated with decreased kinase activity. To determine if this was the case in response to high Pi, mTOR from mouse brain lysate was immunoprecipitated and kinase activity assay was performed with 4E-BP1 phosphorylated heat- and acid-stable (PHAS-1) protein as a substrate. Immunoprecipitation of mTOR was carried out using the Seize Primary Mammalian Immunoprecipitation Kit according to manufacturer's guidelines (Pierce, Rockford, IL, USA). The mTOR kinase assay was performed with 300 μM ATP and 1 μg PHAS-1 (Calbiochem, San Diego, CA) for 30 min at 30°C. Reactions were terminated by adding 5x sample buffer and boiling. The samples were analyzed by 15% SDS/PAG electrophoresis.
Immunohistochemistry.
Formalin-fixed, paraffin-embedded tissue sections (5 μm) were transferred to plus slides (Fisher Scientific, Pittsburgh, PA, USA). The tissue sections were deparaffinized in xylene and rehydrated through alcohol gradients and incubated in 200 μl of proteinase K, then washed and incubated in 0.3% hydrogen peroxide (AppliChem, Darmstadt, Germany) for 30 min to quench endogenous peroxidase activity. After they were in PBS, the tissue sections were incubated with 5% BSA in PBS for 1 h at room temperature to block unspecific binding sites. Primary antibodies were applied on tissue sections overnight at 4°C. The following day, the tissue sections were washed and incubated with secondary HRP-conjugated antibodies for 1 h at room temperature. After careful washing, tissue sections were counterstained with Mayer's hematoxylin (DAKO, Caepinteria, CA, USA) and washed with xylene. Cover slips were mounted using Permount (Fisher, Pittsburgh, PA, USA), and the slides were reviewed in a light microscope (Carl Zeiss, Thornwood, NY, USA).
Data analysis.
Quantification of Western blot analysis was performed using Multi Gauge version 2.02 program (Fujifilm). All results are given as means ± SE. Result were analyzed by Student's t-test (Graphpad Software, San Diego, CA, USA) or two–way analysis of variance (ANOVA; SAS User Guide Statistics, SAS Institute, Cary, NC, USA). A value of p < 0.05 was considered significant (), and p < 0.01 was highly significant () compared to the corresponding control.
RESULTS
High Dietary Levels of Pi Perturb Growth of Cerebellum of Young Mice
The diverse functional roles of Pi and different nutritional requirements during growth suggest that appropriate in vivo nutritional studies at specific stages of growth are needed. Therefore, the potential effects of high levels of Pi on the development of young mice were investigated. A high Pi diet caused reduced body weight after 3 weeks of treatment, although food intake was equivalent to normal food consumption and growth rate was similar (Fig. 1A). A High Pi diet decreased the relative weight of cerebrum slightly; however, the relative weight of cerebellum was significantly decreased (Fig. 1B). A high Pi diet significantly increased the serum phosphate level and significantly decreased calcium concentration as compared to that of the normal diet group (Fig. 1C). Other serum biochemical and hematological values remained within normal range (data not shown).
A High Level of Dietary Pi Increases Brain-Specific NPT1
To confirm the hypothesis that phosphate transport in the brain may play a key role in the homeostasis of brain function as mentioned earlier, the potential effect of high dietary Pi on brain-specific NPT protein expression was evaluated. A high Pi diet significantly increased NPT1 protein expression in both cerebrum and cerebellum (Fig. 2A, B). Densitometric analysis clearly demonstrated how high levels of dietary and serum Pi increased NPT1 protein expression in the test group (Fig. 2C, D).
High Levels of Dietary Pi Regulate Akt Phosphorylation and Akt-Related Signals
Akt is involved in a wide range of important functions in diverse organs, including the brain, and it has been demonstrated to be an important signaling protein in response to nutrient metabolism (Song et al., 2005 and references therein). This fact and the results of our preliminary study, which indicated that high levels of Pi have the potential to change the pattern of Akt protein expression in mouse brains, prompted us to investigate the effects of high levels of Pi on Akt-related signals in vivo. High levels of dietary Pi significantly increased Akt phosphorylation at Ser473 in cerebellum; however, no significant changes were observed in cerebrum. In contrast, high levels of Pi decreased Akt phosphorylation at Thr308 in both cerebrum and cerebellum (Fig. 3A, B). Densitometric analysis (Fig. 3C, D) indicates that for total Akt expression, high dietary Pi decreased protein expression in cerebrum, while that of cerebellum remained unchanged (Fig. 3A, B). In this study, phosphospecific antibodies were employed to measure Akt activation; Akt is activated by phosphorylation (reviewed in Song et al., 2005). To control for loading or changes in total Akt protein the level of Akt activation is represented as a ratio of phosphorylated to total protein. As shown in Figure 3E and F, high levels of Pi increased Akt phosphorylation at Ser473 in both cerebrum and cerebellum but did not affect Akt phosphorylation at Thr308 in cerebrum, although a significant suppression of Akt activity was observed in cerebellum. Such data suggest that high Pi regulates Akt phosphorylation in cerebrum and cerebellum in a somewhat different manner. Our results were further confirmed by immunohistochemical analysis. High levels of Pi decreased the expression of p-Akt (Thr308) compared to that of the normal diet group, especially in cerebellum (Fig. 4A, B). However, high levels of Pi increased the expression of p-Akt (Ser473) in both cerebrum and cerebellum (Fig. 4A, B). To investigate whether high Pi affects Akt-related signal pathways, changes in the appropriate Akt-related signals were measured. High levels of Pi slightly reduced protein expression of mTOR in both regions of the brain, even though there was no statistical significance. In contrast, a change in the expression pattern of the eIF4E-BP1 protein was evident. High levels of Pi significantly decreased eIF4E-BP1 protein expression in cerebrum but did not induce any change in cerebellum (Fig. 5A, B, C, D).
High Dietary Pi Regulate Protein Translation
The data above showing Pi-mediated selective suppression of eIF4E-BP1 in cerebrum strongly suggest that high levels of Pi may regulate cap-dependent protein translation. In fact, recent lines of evidence suggest that cap-dependent protein translation regulation has immense biological significance, and affects such processes as cell growth and development, oncogenic transformation and even the function of the central nervous system (Richter and Sonenberg, 2005). Therefore, the effects of high levels of Pi on protein translation in young mouse brains were closely examined. High levels of Pi slightly increased mTOR kinase activity in cerebrum, but the activity remained unchanged in cerebellum (Fig. 6A, B). A minimal or no change in mTOR kinase activity was observed in cerebrum and cerebellum in response to high levels of Pi, but an increase in cap-dependent protein translation was visible in cerebrum. Firefly luciferase and renilla luciferase assays were performed to determine the levels of cap-independent and cap-dependent protein translation. Results indicated that high levels of Pi enhanced cap-dependent protein translation in cerebrum, more so than in cerebellum, where IRES-mediated protein translation was dominant (Fig. 6C, D).
High Dietary Pi Affects ERK Phosphorylation but Not Mnk Activities
Because ERK is known to negatively regulate eIF4E-BP1 protein expression through Mnk1 (Rolli-Derkinderen et al., 2003; Rosenwald, 2004), and because Akt is closely associated with the ERK pathway (Perkinton et al., 2002), the potential effects of high levels of Pi on ERK and Mnk signals were evaluated. Phosphospecific antibodies were employed to measure the activity of ERK, and suppression of ERK phosphorylation in response to high levels of Pi was observed both in cerebrum and cerebellum. Interestingly, high levels of Pi reduced phosphorylation of ERK1 (p44)—more so than that of ERK2 (p42)—but no significant change was observed in the total ERK1/2 (Fig. 7A, B). High levels of Pi caused a significant reduction in ERK1 phosphorylation over total ERK1 and a slight reduction in ERK2 phosphorylation over total ERK2 in cerebrum (Fig. 7C). A similar pattern was observed in cerebellum. Only ERK1 phosphorylation over total ERK1 was decreased significantly in cerebellum (Fig. 7D). In contrast, Western blot analysis revealed that high levels of Pi did not induce any change in Mnk activity (data not shown).
High Levels of Pi Decreased Cell Proliferation through Suppression of Cyclin D1 and PCNA
Inorganic phosphorous is known as a signaling molecule capable of regulating global gene expression and cellular functioning in multiple cell types (Beck et al., 2003). Therefore, this study was undertaken to evaluate the effects of high levels of Pi on brain cell proliferation, which was investigated by Western blot analysis, measuring the expression of marker proteins characteristic of cell cycle such as PCNA and cyclin D1. The study found that high levels of Pi caused a significant suppression of the protein expression of both PCNA and cyclin D1 in both regions of the brain (Fig. 8A, B, C, D).
DISCUSSION
Serum Pi level is maintained within a narrow range through complex interaction between intestinal absorption, exchange with intracellular and bone storage pools, and renal tubular reabsorption. As mentioned earlier, Pi plays a key role in diverse physiological functions. Recently, the NPT specific to the brain has been identified, and considerable progress has been made in our understanding of their function and regulation (Hayashi et al., 2001; Hisano, 2002). Pi transport into the brain is regulated mainly by dietary and serum Pi (Takeda et al., 2004). Research indicates that Pi works as a stimulus capable of increasing or decreasing the expression of a number of pivotal genes, such as those involved in the regulation of transcription, signal transduction, and cell cycle (Beck, 2003). It is thought that an excess of Pi intake over a long period of time is a strong factor in aging (Takeda et al., 2004). Therefore, restricting Pi consumption seems to be important in maintaining good quality of life. These reasons, the potential importance of Pi as a novel signaling molecule, and the risk of diverse neurodegenerative diseases that involve brain-specific NPT1, prompted us to define the pathways by which Pi affects the growth of young mouse brain.
In this study, the correlation between growth and high levels of Pi exposure during the development of weanling mice was examined. The high Pi diet group demonstrated reduced body weight (Fig. 1A) in addition to decreased relative brain weight (Fig. 1B), suggesting the importance of phosphate levels in specific tissues and in overall normal growth. The AIN93 diet composition itself used in this study should not affect the animals' growth, because its composition fulfills the requirements for normal growth in laboratory animals; thus we can exclude the possibility of malnutrition (Reeves et al., 1993) and consider the possibility that factors other than malnutrition could be critical for causing the growth retardation. For example, high serum phosphate levels could contribute to the development of arteriosclerosis and could lead to secondary hyperparathyroidism, both of which are detrimental to health and hinder normal development (Sitara et al., 2004). Significant reductions in serum Ca2+ levels in response to high levels of Pi may also shed light on the detrimental effects of high levels of Pi on normal growth (Fig. 1C). In fact, calcium is responsible for the tight regulation of the secretion of parathyroid hormone (PTH), an excess of which results in hyperparathyroidism. To maintain an appropriate level of PTH, its degradation normally occurs within the parathyroid gland prior to secretion (Shoback et al., 2003). In this study, we hypothesize that high Pi–induced reduction of serum Ca2+ interferes with normal degradation of PTH, and in turn retards growth. This hypothesis is supported by a recent study that demonstrated how low concentrations of extracellular Ca2+ downregulate degradation of PTH through the suppression of (1–84)hPTH generation, which is responsible for N-terminal truncation of PTH in parathyroid cells (Kawata et al., 2005). It can therefore be hypothesized that high Pi–induced reductions of serum Ca2+ levels may lead to hyperparathyroidism, which interferes with normal growth through bone resorption by impairing degradation of PTH.
Akt is a potent molecule that has been shown to be necessary for the survival of a variety of neurons and non-neuronal cells. A recent study has also demonstrated that adhesion molecules on glia play an important role in the regulation of brain growth through Akt phosphorylation (Scheidenhelm et al., 2005). Both of these facts suggest that a disruption in Akt activity may have an adverse effect on normal development of the brain. Akt is activated by phosphorylation at two important sites, Thr308 and Ser473 (Kandel and Hay, 1999). Phosphorylation at both sites is required for full activity, and suppression of phosphorylation at either site can induce abrogation of tumor development (Kim et al., 2004). Results of our study show an increase in high Pi-induced Akt phosphorylation at Ser473 in both cerebrum and cerebellum, but a decrease in phosphorylation at Thr308 in cerebellum (Fig. 3E, F). In addition, other data show that reduced activity of Akt due to an altered pattern of phosphorylation is coupled with a significant decrease in protein expressions of PCNA and cyclin D1 (Fig. 8). These results strongly suggest that high levels of Pi may affect brain growth by disturbing Akt activity through different modes of action in cerebrum and cerebellum. Recent evidence indicates that the Akt family has three highly conserved proteins as members, namely, Akt1, Akt2, and Akt3, the first two of which are required for normal growth and metabolism, respectively (Easton et al., 2005). Akt1, required for normal growth, was used in this study, and decreased Akt activity in response to high levels of Pi seemed to disturb the proliferation of brain cells somewhat differently in cerebrum and cerebellum. This correlation has also been noted in research conducted by Easton et al., where Akt1- and Akt3-deficient brains were reduced in size by approximately the same amount. Furthermore, the absence of Akt1 led to a reduction in cell numbers, whereas the lack of Akt3 resulted in smaller and fewer cells. Additionally, attenuated mTOR signaling was observed in the brains of Akt3-deficient mice but not in Akt1-deficient mouse brains (Easton et al., 2005).
Our results also indicate that high levels of dietary Pi do not have a significant effect on the levels of mTOR in either region of the mouse brain were Akt phosphorylation is downregulated (Fig. 5). Therefore, high levels of Pi may affect cell proliferation, not through Akt1, but through other types of Akt. In effect, high levels of Pi may downregulate brain cell growth through disturbed Akt activity; however, the mode of action in cerebrum may be somewhat different from that of cerebellum. Therefore, the mechanisms by which high Pi induces the disruption of Akt activity in cerebrum and cerebellum should be further elucidated, and extensive efforts to reveal such mechanisms are under way.
As discussed previously, Akt signaling is closely associated with the ERK pathway (Rosenwald, 2004). Tests were performed in our study to examine whether high levels of Pi affect ERK and Mnk1 in the brain. Results indicate that in response to high dietary levels of Pi, ERK1 activation is significantly downregulated in both cerebrum and cerebellum, whereas no significant change in ERK2 activation was observed (Fig. 7C, D). It has been demonstrated that transgenic mice overexpressing phosphorylated ERK1/2 in the brain are protected against oxygen neurotoxicity (Felderhoff-Mueser et al., 2004). Such activated ERK may be partially responsible for the protection of brain cells against oxidative stress. In our study, high dietary Pi suppressed ERK activation, especially ERK1. This suggests that long-term exposure to excess dietary Pi may increase the susceptibility of the young mouse brain to xenobiotic stimuli.
The regulation of cyclin D1 expression can occur at many levels (Perkinton et al., 2002), but whether expression of cyclin D1 is regulated at a step that can be strictly defined as a translation stage, has not been determined. Expression of cyclin D1 can be induced independently either through the PI3K (Takuwa et al., 1999) or the ERK (Lavoie et al., 1996) pathway. However, a recent study has revealed crosstalk between Akt and the ERK signals in cultured rat cerebellar granule cells (Myhre et al., 2004), suggesting that the suppressed expression of cyclin D1 in cerebrum and cerebellum in our study could also result from crosstalk between Akt and ERK signals. In fact, reduced phosphorylation of Thr308 and Ser473 of Akt (Fig. 3E, F), and the selective suppression of ERK1 phosphorylation (Fig. 7C, D) that occurred simultaneously, may represent a mechanism to explain the impairment of brain cell growth in our study. Put succinctly, high levels of Pi may increase the susceptibility of brain cells to external stimuli through crosstalk between Akt and ERK, and reduced serum Ca2+ levels may aggravate the process.
It was observed in the present study that high Pi–stimulated cap-dependent translation occurs in cerebrum, whereas cap-independent translation takes place in cerebellum (Fig. 6C, D). Selective augmentation of Akt phosphorylation at Ser473 in cerebrum (Fig. 3E) and an increase in Akt phosphorylation at Ser473 coupled with a decrease in Akt phosphorylation at Thr308 in cerebellum (Fig. 3F), may be partially responsible for the differential translation. In fact, differential regulation of cap-independent translation and decreased cyclin D1 expression may account for the differential sensitivity of high-vs-low Akt activity (Shi et al., 2005).
Current research suggests that cyclin D1 is normally accumulated during the G2 phase of the cell cycle and that synthesis during this phase may factor into the rapid rise in cyclin D1 to the level required to initiate G1 transit in actively proliferating cells (Sherr, 2002). The reduction in cap-dependent protein synthesis during the G2/M cell cycle transition has also been recently underscored (Pyronnet et al., 2001). Our data imply that Akt-related cyclin D1 reduction is closely associated with ERK inactivation. Experiments designed to elaborate on this implication are under way. Our results support a working model in which Akt-dependent control of cyclin D1 can regulate the expression of critical determinants for G1 arrest or aging.
In summary, the present study suggests that high levels of dietary Pi may have detrimental effects on the normal growth of young mouse brains through the disruption of Akt-ERK signals. The Akt-ERK signaling pathway may also be involved in numerous biological processes critical to normal development and may modulate various neurodegenerative diseases of the brain. Additionally, elevated dietary Pi caused a shift in cap-dependent verse cap-independent translation, which also may negatively affect tissue function. Extensive studies to elucidate the precise effects of such activated signals on both development and pathogenesis in the brain are in progress.
ACKNOWLEDGMENTS
This work was supported by grants from the Korea Science and Engineering Foundation (KOSEF). J.H., S.K.H., and K.Y. are also grateful for the award of the BK21 fellowship. K.H.L. was supported by the grants from 21C Frontier Functional Human Genome Project and National Nuclear R&D Program from Ministry of Science and Technology GRB. Jr. was supported by a National Cancer Institute Grant (CA084573).
REFERENCES
Bauer, M., Alda, M., Priller, J., and Young, L. T. The International Group for the Study of Lithium Treated Patients (IGSLI). (2003). Implications of the neuroprotective effects of lithium for the treatment of bipolar and neurodegenerative disorders. Pharmacopsychiatry 36(Suppl. 3), S250–S254.
Beck, G. R., Jr. (2003). Inorganic phosphate as a signaling molecule in osteoblast differentiation. J. Cell. Biochem. 90, 234–243.
Beck, G. R., Moran, E., and Knecht, N. (2003). Inorganic phosphate regulates multiple genes during osteoblast differentiation, including Nrf2. Exp.Cell Res. 288, 288–300.
Bryan, J., Osendarp, S., Hughes, D., Calvaresi, E., Baghurst, K., and van Klinken, J. W. (2004). Nutrients for cognitive development in school-aged children. Nutr. Rev. 62, 295–306.
Brywe, K. G., Mallard, C., Gustavsson, M., Hedtjarn, M., Leverin, A. L., Wang, X., Blomgren, K., Isgaard, J., and Hagberg, H. (2005). IGF-I neuroprotection in the immature brain after hypoxia–ischemia, involvement of Akt and GSK2beta Eur. J. Neurosci. 21, 1489–1502.
Calvo, M. S. (1993). Dietary phosphorous, calcium metabolism, and bone. J. Nutr. 123, F1627–F1633.
Caverzasio, J., and Bonjour, J. P. (1996). Characteristics and regulation of Pi transport in estrogenic cells for bone metabolism. Kidney Int. 49, 975–980.
Conrads, K. A., Yi, M., Simpson, K. A., Lucas, D. A., Camalier, C. E., Yu, L. R., Veenstra, T. D., Stephens, R. M., Conrads, T. P., and Beck, G. R., Jr. (2005). A combined proteome and microarray investigation of inorganic phosphate-induced pre-osteoblast cells. Mol. Cell Proteomics 9, 1284–1296.
Cote, A., Chiasson, M., Perelta, M. R., 3rd, Lafortune, K., Pellegrini, L., and Toth, K. (2005). Cell type–specific action of seizure-induced intracellular zinc communication in the rat hippocampus. J. Physiol. 566, 821–837.
Creancier, L., Mercier, P., Prats, A., and Morello, D. (2001). C-myc internal ribosome entry site activity is developmentally controlled and subjected to a strong translational repression in adult transgenic mice. Mol. Cell. Biol. 21, 1833–1840.
Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., and Hemmings, B. A. (1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378, 785–789.
Easton, R. M., Cho, H., Roovers, K., Shineman, D. W., Mizrahi, M., Forman, M. S., Lee, V. M., Szabolcs, M., de Jong, R., Oltersdorf, T., et al. (2005). Role of Akt3/protein kinase B gamma in attainment of normal brain size. Mol. Cell. Biol. 25, 1869–1878.
Felderhoff-Mueser, U., Bittigau, P., Sifringer, M., Jarosz, B., Korowicz, E., Mahler, L., Piening, T., Moysich, A., Grune, T., Thor, F., et al. (2004). Oxygen causes cell death in the developing brain. Neurobiol. Dis. 17, 273–282.
Glinn, M., Ni, B., and Paul, S. M. (1995). Characterization of Na(+)-dependent phosphate uptake in cultured fetal rat cortical neurons. J. Neurochem. 65, 2358–2365.
Hayashi, M., Otsuka, M., Morimoto, R., Hirota, S., Yatsushiro, S., Takeda, J., Yamamoto, A., and Moriyama, Y. (2001). Differentiation-associated Na+-dependent inorganic phosphate cotransporter (DNPI) is a vesicular glutamate transporter in endocrine glutamatergic systems. J. Biol. Chem. 276, 43400–43406.
Hisano, S. (2002). The inorganic phosphate transport system in the brain and the potency as vesicular glutamate transporters. Kaibogaku Zasshi 77, 51–58.
Hovelmann, S., Beckers, T. L., and Schmidt, M. (2004). Molecular alterations in apoptotic pathways after PKB/Akt-mediated chemoresistance in NCI H460 cells. Br. J. Cancer 90, 2370–2377.
Kandel, E. S., and Hay, N. (1999). The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB. Exp. Cell Res. 253, 210–229.
Kane, L. P., Mollenauer, M. N., Xu, Z., Turck, C. W., and Weiss, A. (2002). Akt-dependent phosphorylation specifically regulates Cot induction of NF-kappa B-dependent transcription. Mol. Cell. Biol. 22, 5962–5974.
Kawata, T., Imanishi, Y., Konayashi, K., Onoda, N., Takemoto, Y., Tahara, H., Okuno, S., Ishimura, E., Miki, T., Ishikawa, T., et al. (2005). Direct in vitro evidence of extracellular Ca2+ induced N-terminal truncation of (1–84)hPTH by human parathyroid cells. J. Clin. Endocrinol. Metab. 90, 5774–5778.
Kim, H. W., Park, I. K., Cho, C. S., Lee, K. H., Beck, G. R., Colburn, N. H., and Cho, M. H. (2004). Aerosol gene delivery of glucosylated polyethyleneimine (GPEI)/PTEN complex suppresses Akt downstream pathways in the lung of K-ras null mice. Cancer Res. 64, 7971–7976.
LaManna, J. C., and Lust, W. D. (1997). Nutrient consumption and metabolic perturbations. Neurosurg Clin North Am. 8, 145–163.
Lavoie, J. N., L'Allemain, G., Brunet, A., Muller, R., and Pouyssegur, J. (1996). Cyclin D1 expression is regulated positively by the p42/p44 MAPK and negatively by the p38/HOGMAPK pathway. J. Biol. Chem. 271, 20608–20616.
Lawlor, M. A., and Alessi, D. R. (2001). PKB/Akt: A key mediator of cell proliferation, survival and insulin responses. J. Cell Sci. 114, 2903–2910.
Mulroney, S. E., Woda, C. B., Halaihel, N., Louie, B., McDonnell, K., Schulkin, J., Haramati, A., and Levi, M. (2004). Central control of renal sodium-phosphate (NaPi-2) transporters. Am. J. Physiol. Renal Cell Physiol. 286, F647–F652.
Myhre, O., Sterri, S. H., Bogen, I. L., and Fonnum, F. (2004). ERK1/2 phosphorylation and reactive oxygen species formation via nitric oxide and Akt-1/Raf-1 crosstalk in cultured cerebellar granule cells exposed to the organic solvent 1,2,4-trimethylcyclohexane. Toxicol.Sci. 80, 296–303.
Ni, B., Du, Y., Wu, X., DeHoff, B. S., Rostek, P. R., and Paul, S. M. (1996). Molecular cloning, expression, and chromosomal localization of a human brain-specific Na+-dependent inorganic phosphate cotransporter. J. Neurochem. 66, 2227–2238.
Onyango, I. G., Bennett, J. P., Jr., and Tuttle, J. B. (2005). Endogenous oxidative stress in sporatic Alzheimer/s disease neuronal cybrids reduces viability by increasing apoptosis through pro-death signaling pathways and is mimicked by oxidant exposure of control cybrids. Neurobiol. Dis. 19, 312–322.
Perkinton, M. S., Ip, J. K., Wood, G. L., Crossthwaite, A. J., and Williams, R. J. (2002). Phosphatidylinositol 3-kinase is a central mediator of NMDA receptor signaling to MAP kinase (ERK1/2), Akt/PKB and CREB in striatal neurons. J. Neurochem. 80, 239–254.
Pollitt, E., Jacoby, E., and Cueto, S. (1996). School breakfast and cognition among nutritionally at-risk children in the Peruvian Andes. Nutr. Rev. 54, S22–S26.
Pyronnet, S., Dosite, J., and Sonnenberg, N. (2001). Suppression of cap-dependent translation in mitosis. Genes Dev. 15, 2083–2093.
Reeves, P. G., Nielsen, F. H., and Fahey, G. C., Jr. (1993). AIN-93 purified diets for laboratory rodents: Final report of the American Institute of Nutrition ad hoc Writing Committee on the Reformulation of the AIN-76A rodent diet. J. Nutr. 123, 1939–1951.
Reinert, M., Khaldi, A., Zauner, A., Doppenberg, E., Choi, S., and Bullock, R. (2000). High level of extracellular potassium and its correlates after severe head injury: Relationship to high intracranial pressure. J. Neurosurg. 93, 800–807.
Richter, J. D., and Sonenberg, N. (2005). Regulation of cap-dependent translation by eIF4E inhibitory proteins. Nature 433, 477–480.
Rolli-Derkinderen, M., Machavoine, F., Baraban, J. M., Grolleau, A., Beretta, L., and Dy, M. (2003). ERK and p38 inhibit the expression of 4E-BP1 repressor of translation through induction of Egr-1. J. Biol. Chem. 278, 18859–18867.
Rosenwald, I. B. (2004). The role of translation in neoplastic transformation from a pathologist's point of view. Oncogene 23, 3230–3237.
Scheidenhelm, D. K., Cresswell, J., Haipek, C. A., Fleming, T. P., Mercer, R. W., and Gutmann, D. H. (2005). Akt-dependent cell size regulation by the adhesion molecule on glia occurs independently of phosphatidylinositol-3-kinase and Rheb signaling. Mol. Cell. Biol. 25, 3151–3162.
Sherr, C. J. (2002). D1 in G2. Cell Cycle 1, 36–38.
Shi, Y., Sharma, A., Wu, H., Lichtenstein, A., and Gera, J. (2005). Cyclin D1 and c-myc internal ribosome entry site (IRES)-dependent translation is regulated by Akt activity and enhanced by rapamycin through a p38 MAPK and ERK-dependent pathway. J. Biol. Chem. 280, 10964–10973.
Shoback, D. M., Bilezikian, J. P., Turner, S. A., McCary, L. C., Guo, M. D., and Peacock, M. (2003). The calcimimetic cinacalcet normalizes serum calcium in subjects with primary hyperthyroidism. J. Clin. Endocrinol. Metab. 88, 5644–5649.
Sitara, D., Razzaque, M. S., Hesse, M., Yoganathan, S., Taguchi, T., Erben, R. G., Juppner, H., and Lanske, B. (2004). Homozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverses hypophosphatemia in Phex-deficient mice. Matrix Biol. 23, 421–432.
Song, G., Ouyang, G., and Bao, S. (2005). The activation of Akt/PKB signaling pathway and cell survival. J. Cell Mol. Med. 9, 59–71.
Takeda, E., Yamamoto, H., Nashiki, K., Sato, T., Arai, H., and Taketani, Y. (2004). Inorganic phosphate homeostasis and the role of dietary phosphorus. J. Cell. Mol. Med. 8, 191–200.
Takuwa, N., Fukui, Y., and Takuwa, Y. (1999). Cyclin D1 expression mediated by phosphatidylinositol-3-kinase through mTOR-p70S6K-independent signaling in growth factor-stimulated NIH3T3 fibroblasts. Mol. Cell. Biol. 19, 1346–1358.
Uluitu, M., Badescu, A., and Chis, R. (1995). Influence of increased sodium intake on cerebral excitability. Rom. J. Physiol. 32, 11–33.(Hua Jin, Soon-Kyung Hwang, Kyungnam Yu, )