Phornphutkul C, Wu KY, Auyeung V, Chen Q, Gruppuso PA.

Source

Department of Pediatrics, Division of Pediatric Endocrinology and Metabolism, Rhode Island Hospital and Brown University, Providence, Rhode Island 02903, USA. chanika_phornphutkul@brown.edu

Abstract

The mammalian Target Of Rapamycin (mTOR) is a nutrient-sensing protein kinase that regulates numerous cellular processes. Fetal rat metatarsal explants were used as a physiological model to study the effect of mTOR inhibition on chondrogenesis. Insulin significantly enhanced their growth. Rapamycin significantly diminished this response to insulin through a selective effect on the hypertrophic zone. Cell proliferation (bromodeoxyuridine incorporation) was unaffected by rapamycin. Similar observations were made when rapamycin was injected to embryonic day (E) 19 fetal rats in situ. In the ATDC5 chondrogenic cell line, rapamycin inhibited proteoglycan accumulation and collagen X expression. Rapamycin decreased content of Indian Hedgehog (Ihh), a regulator of chondrocyte differentiation. Addition of Ihh to culture medium reversed the effect of rapamycin. We conclude that modulation of mTOR signaling contributes to chondrocyte differentiation, perhaps through its ability to regulate Ihh. Our findings support the hypothesis that nutrients, acting through mTOR, directly influence chondrocyte differentiation and long bone growth.

Me: It seems that mTOR may be one of the things that regulates Ihh.

1. Mirei Murakami1,2, Tomoko Ichisaka1,2, Mitsuyo Maeda3, Noriko Oshiro2,4,Kenta Hara2,5, Frank Edenhofer6, Hiroshi Kiyama3, Kazuyoshi Yonezawa2,4,* and Shinya Yamanaka1,2,*

+Author Affiliations

• ¹Research and Education Center for Genetic Information, Nara Institute of Science and Technology
• ²CREST, Japan Science and Technology Agency, Nara 630-0192
• ³Department of Anatomy and Neurobiology, Osaka City University Medical School, Osaka 545-8585
• ⁴Biosignal Research Institute, Kobe University, Hyogo 657-8501
• ⁵Fourth Department of Internal Medicine, Kobe University School of Medicine, Hyogo 650-0017, Japan
• ⁶Institute of Reconstructive Neurobiology, University of Bonn Medical Center, D-53105 Bonn, Germany

ABSTRACT

TOR is a serine-threonine kinase that was originally identified as a target of rapamycin in Saccharomyces cerevisiae and then found to be highly conserved among eukaryotes. In Drosophila melanogaster, inactivation of TOR or its substrate, S6 kinase, results in reduced cell size and embryonic lethality, indicating a critical role for the TOR pathway in cell growth control. However, the in vivo functions of mammalian TOR (mTOR) remain unclear. In this study, we disrupted the kinase domain of mouse mTOR by homologous recombination. While heterozygous mutant mice were normal and fertile, homozygous mutant embryos died shortly after implantation due to impaired cell proliferation in both embryonic and extraembryonic compartments. Homozygous blastocysts looked normal, but their inner cell mass and trophoblast failed to proliferate in vitro. Deletion of the C-terminal six amino acids of mTOR, which are essential for kinase activity, resulted in reduced cell size and proliferation arrest in embryonic stem cells. These data show that mTOR controls both cell size and proliferation in early mouse embryos and embryonic stem cells.

Me: So mTOR in general controls cell size and proliferation in stem cells and embryos. That sounds pretty important in longitudinal growth to me.

From link HERE

mTOR inhibitors

mTOR is a kinase protein. It can make cells produce chemicals such as cyclins that trigger cell growth. It may also trigger cells to produce proteins which trigger the development of new blood vessels that cancers need in order to grow. In some types of cancer mTOR is switched on, which makes the cancer cells grow and produce new blood vessels. mTOR inhibitors are a new type of cancer growth blocker being used to try to stop the growth of some cancers. mTOR inhibitors include temsirolimus (Torisel), everolimus (Afinitor) and deforolimus.

As for Leucine, from link HERE

Leucine restriction inhibits chondrocyte proliferation and differentiation through mechanisms both dependent and independent of mTOR signaling

UNDERNUTRITION IS A WELL-DOCUMENTED cause of poor linear growth, whereas obesity produces accelerated linear growth in children. Primary causes of undernutrition are complex. They can range from inadequate availability of calories to inadequate specific nutrients, such as protein or amino acids. Despite the many causes and forms of undernutrition, one universal outcome is poor long bone growth, which can be presumed to be an effect on endochondral bone elongation (1, 6). Over the last several decades, the mechanisms by which nutritional status affects bone growth have focused on indirect effects via changes in insulin-like growth factor I (IGF-I) biological effect (3, 7). Impaired IGF-I production in undernutrition is in part associated with impaired hepatic growth hormone (GH) sensitivity, which is associated with decreased hepatic GH receptor expression, hepatic IGF-I mRNA, and circulating IGF-I levels (31). Similarly, overnutrition is associated with upregulation of the GH/IGF-I axis (23).

The effect of IGF-I on chondrocyte growth and differentiation within the growth plate has been well documented (14, 24). We and others have demonstrated that IGF-I has an important role in chondrocyte proliferation and differentiation (25, 28). In addition to the effects of IGF-I on chondrocytes, we have demonstrated that insulin at physiological concentration has a direct effect on chondrocyte differentiation (27), thus providing for another mechanism by which nutritional status can modulate bone growth.

In recent years, mechanisms by which nutrients exert a direct effect on cell growth and function have been elucidated (17). Although restriction of essential amino acids has been viewed as limiting because of their requirement as substrates for protein synthesis, essential amino acids also act as signaling factors in several regulatory pathways (15,16). Perhaps the most well-characterized signaling pathway regulated by amino acids has at its center the mammalian target of rapamycin (mTOR) (17). mTOR is a nutrient-sensing kinase that integrates input from amino acids, growth factors, and the energy status of the cell (15). It acts as a central controller of translation, controlling ribosomal biogenesis and global protein synthesis (4). The TOR protein is highly conserved from yeast to mammals (5). Rapamycin, a widely used immunosuppressive agent, directly inhibits mTOR activity and has been key to understanding the role of mTOR in cell regulation (22, 35).

We have recently demonstrated the effect of rapamycin on chondrocyte growth and differentiation in ATDC5 cells (26), fetal rat metatarsal explants (26), and the rabbit growth plate (unpublished observation). mTOR inhibition with rapamycin results in significantly decreased chondrocyte differentiation, a modest decrease in chondrocyte proliferation, and decreased total bone growth in physiological systems.

Leucine is the most potent nutrient regulator of mTOR signaling (29, 30). We therefore hypothesized that leucine availability would have a direct effect on chondrocyte growth and differentiation, resulting in decreased longitudinal bone growth when restricted. We further hypothesized that the effect of leucine restriction would be a direct result of mTOR inhibition, although mTOR-independent pathways have been described. One of these involves the mammalian general control nonderepressible 2 (GCN2) kinase (32). GCN2 is a stress kinase that is activated by amino acid starvation to modulate protein synthesis. GCN2 phosphorylates the α-subunit of the eukaryotic initiation factor (eIF2). The response that allows organisms to tolerate amino acid deprivation in states of malnutrition and starvation involves repression of protein synthesis and upregulation of amino acid biosynthesis and transport (10) with a net effect of decreased cell growth.

Based on our hypothesis and well-defined mechanisms that control chondrocyte growth and differentiation, we have performed studies using embryonic day 19 (E19) fetal rat metatarsal explants. The benefit of this model is the intact bone maintains its cell-cell and cell-matrix interaction. We have also used the ATDC5 chondrogenic cell line to extend our observations to an in vitro model. Last, using microarray analysis, we have explored other potential mechanisms accounting for the effect of leucine restriction on chondrocyte growth and differentiation.

DISCUSSION

The present studies were aimed at characterizing the effect of restricting a key nutrient, leucine, on two models of bone growth: metatarsal explants and growth and a chondrogenic cell line. Amino acids, particularly the branched-chain amino acids, regulate protein synthesis beyond the level of their own availability as the substrate for peptide-chain elongation (15, 17). They do so by functioning as signaling molecules. Leucine appears to be the most potent of the branched-chain amino acids in this regard, having a potent effect on signaling via two important signaling kinases, mTOR and GCN2 (8, 10,32, 33).

The mTOR signaling pathway has been shown to mediate the effects of leucine on mRNA translation initiation. We recently demonstrated the important role of mTOR in the regulation of chondrocyte growth and differentiation (26). Our previous findings provided for a mechanism whereby nutrients, acting through mTOR, can directly modify linear growth. In the present study, we performed analogous experiments comparing the effect of leucine restriction in the fetal metatarsal explant model as well as ATDC5 cells.

The present studies were undertaken to test the hypothesis that leucine restriction would exert its effects on bone growth through its ability to signal via mTOR. Using the more physiological bone explant model, we confirmed that leucine affected growth in a dose-dependent manner. The observed growth inhibition was associated with decreased chondrocyte proliferation as measured by decreased BrdU incorporation. Decreased chondrocyte proliferation presumably results in fewer chondrocytes that are available to differentiate and become hypertrophic cells, consistent with the decreased hypertrophic zone height that we observed.

Chondrocytes that differentiate into prehypertrophic and hypertrophic chondrocytes undergo a 4- to 10-fold increase in cytoplasmic volume, making distal hypertrophic cells a significant component of longitudinal bone growth. In rapidly growing bones, ∼10% of bone length is contributed by proliferating cells, a one-third by matrix synthesis throughout the growth plate, and nearly two-thirds by the contribution of hypertrophic cells (34).

Although leucine restriction appears to have an effect on chondrocyte proliferation and differentiation, we did not observe an increase in apoptosis as assessed by TUNEL staining. Premature cell death can result in reduced bone growth, as observed in humans with skeletal dysplasia (12) and in mice with disruption of genes important to chondrogenesis, including those encoding filamin B, matrilin-3, or components of the β-catenin signaling pathway (11, 21). Our results indicate that the effect of leucine restriction occurs during the early phase of the chondrocyte growth and differentiation process, not as a result of increased apoptosis.

Because mTOR is a signaling target for leucine, we examined the effect of leucine restriction on mTOR signaling in metatarsals by assessing the phosphorylation state of ribosomal protein S6. We observed only a modest decrease in phospho-S6 staining in the explants grown in 0.02 mM leucine. This was in sharp contrast to rapamycin, which abolished phospho-S6 staining. This raised the possibility that leucine response was mediated through a mechanism other than one involving modulation of mTOR activity.

Using the ATDC5 chondrogenic cell line, we extended our observations in an attempt to support or refute conclusions drawn from the metatarsal explant studies. Leucine restriction decreased chondrocyte differentiation as measured by accumulation of proteoglycan and expression of collagen X. Total cell mass measured by Neutral Red accumulation was also decreased. Again, markers of mTOR activity, S6 phosphorylation and the pattern of 4E-BP1 phosphorylation, were modestly inhibited under conditions of leucine restriction relative to the marked effect of rapamycin. The expression of Ihh, a key contributor to chondrocyte proliferation and differentiation (9, 18, 20), was also decreased under conditions of leucine restriction. We previously demonstrated that mTOR inhibition may directly regulate Ihh expression (26).

In an effort to identify an mTOR-independent pathway to account for the effects of leucine restriction, we examined the regulation of GCN2 in the ATDC5 cell line. The GCN2 pathway is activated by the accumulation of uncharged tRNAs during amino acid starvation as shown in myoblasts (10). This leads to the phosphorylation of eIF2α, resulting in inhibition of translation initiation of cellular proteins and a global reduction in protein synthesis (10, 13). We observed only a modest increase in the phosphorylation of eIF2α under conditions of leucine restriction.

Microarray analysis of the effects of leucine restriction vs. rapamycin on gene expression in ATDC5 cells revealed that only a small proportion of genes was affected by both leucine restriction and rapamycin. These genes, which numbered 176, accounted for 11.2% of the genes that were affected in leucine restriction. The large effect of leucine restriction relative to rapamycin (1,571 vs. 535 genes) may indicate that leucine restriction has a broader effect on chondrocyte growth and differentiation than does targeted inhibition of mTOR. Using very stringent criteria for significance, gene ontology and pathway analysis further supported marked differences in the effects of the two conditions.

In summary, our studies show that leucine restriction affects proliferation and differentiation in the ATDC5 chondrogenic cell line. Results using the fetal metatarsal explant model are consistent with the ATDC5 studies. Our findings support the conclusion that both mTOR and GCN2 signaling may contribute to the effect of leucine restriction on chondrocyte proliferation and differentiation and, therefore, on long bone growth. However, our studies are also consistent with the possibility that the effects of leucine restriction are mediated by pathways that are independent of effects on these two signaling kinases. We are left to conclude that the mechanism by which leucine restriction inhibits chondrogenesis and attenuates linear bone growth is complex, likely involving modulation of multiple pathways that may involve mTOR and GCN2, but that may also be independent of both of these well-characterized pathways.