SLC1A5 provides glutamine and asparagine necessary for bone development in mice
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Evaluation Summary:
The authors report that intracellular transport of glutamine and asparagine is critical for osteoblast anabolism. The authors use a variety of in vivo and in vitro assays for the testing of their working hypothesis. The paper expands and deepens our knowledge of the role of cellular metabolism in osteoblast function and bone development.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Osteoblast differentiation is sequentially characterized by high rates of proliferation followed by increased protein and matrix synthesis, processes that require substantial amino acid acquisition and production. How osteoblasts obtain or maintain intracellular amino acid production is poorly understood. Here, we identify SLC1A5 as a critical amino acid transporter during bone development. Using a genetic and metabolomic approach, we show SLC1A5 acts cell autonomously to regulate protein synthesis and osteoblast differentiation. SLC1A5 provides both glutamine and asparagine which are essential for osteoblast differentiation. Mechanistically, glutamine and to a lesser extent asparagine support amino acid biosynthesis. Thus, osteoblasts depend on Slc1a5 to provide glutamine and asparagine, which are subsequently used to produce non-essential amino acids and support osteoblast differentiation and bone development.
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Author Response
Reviewer #2 (Public Review): Osteoblasts are highly anabolic cells that display a high proliferation rate and secrete ample amounts of extracellular matrix, indicating that these cells have a specific metabolic profile. Here, using a set of in vivo and in vitro experiments, Sharma et al describe that SLC1A5-mediated glutamine and asparagine uptake is critical to sustain osteoblast anabolism. While the experimental setup is robust, this concept has already been put forth, questioning therefore the novelty of the results. In addition, some of the author's claims are insufficiently supported by the presented data. Especially the metabolic role of asparagine in regulating osteoblast differentiation remains enigmatic. The main concerns are detailed below.
- Based on their data, the authors propose that the main mechanism …
Author Response
Reviewer #2 (Public Review): Osteoblasts are highly anabolic cells that display a high proliferation rate and secrete ample amounts of extracellular matrix, indicating that these cells have a specific metabolic profile. Here, using a set of in vivo and in vitro experiments, Sharma et al describe that SLC1A5-mediated glutamine and asparagine uptake is critical to sustain osteoblast anabolism. While the experimental setup is robust, this concept has already been put forth, questioning therefore the novelty of the results. In addition, some of the author's claims are insufficiently supported by the presented data. Especially the metabolic role of asparagine in regulating osteoblast differentiation remains enigmatic. The main concerns are detailed below.
- Based on their data, the authors propose that the main mechanism whereby SLC1A5 regulates osteoblast proliferation and differentiation is via glutamine uptake, while asparagine only contributes to a lesser extent. Importantly, the concept that glutamine metabolism regulates proliferation and differentiation of osteogenic cells by sustaining anabolic processes has already been described recently, even by the same research group (Yu Y. Cell Metab. 2019; Stegen S. JBMR 2021), questioning the novelty of the present study. Moreover, no metabolic rescue experiments were performed to unequivocally demonstrate that the defect in amino acid/protein synthesis in SLC1A5-deficient cells was causing the decrease in osteoblast proliferation and differentiation.
We appreciate the reviewer’s thorough and thoughtful review and we thank the reviewer for helping us to improve this manuscript. To address this, we evaluated proliferation or osteoblast marker genes in Slc1a5 deficient cells cultured in media supplemented with 10 times the normal concentration of the reduced amino acids (excluding Gln and Asn, Fig. 4B). There was no effect on EDU incorporation, however exogenous amino acids did rescue the induction of Ibsp and Bglap to a lesser extent (Fig. S6D-E). Interpretation of these types of experiments are tricky as the uptake of NEAA may be inherently limited in osteoblasts and due to time constraints, we were unable to quantify intracellular amino acid levels in the rescued cells. Regardless, we interpret these data as affirming the necessity of Slc1a5 to provide Gln and Asn used to synthesize amino acids for osteoblast differentiation. In addition, these data indicate other metabolites (e.g. alpha-ketoglutarate, glutathione, nucleotides etc) derived from Gln and/or Asn are required for proliferation. We have modified the discussion to address this uncertainty.
In addition, Gln and Asn tracing (carbon and nitrogen) in SLC1A5-deficient cells would confirm that Gln and Asn uptake via SLC1A5 is important for osteoblast functioning.
We did not perform tracing experiments in the Slc1a5 deficient cells. We directly evaluated amino acid uptake using radiolabeled amino acids in Slc1a5 deficient cells (Figure 4). Slc1a5 ablation reduced the uptake of Gln and Asn. To test if Gln and Asn uptake was important for osteoblast function we directly compared the cellular effects of Slc1a5 ablation to Gln or Asn withdrawal. From these experiments we concluded that Gln and Asn uptake is essential for osteoblast differentiation.
- Using isotopic labeling experiments, the authors demonstrate that asparagine-derived carbon and nitrogen label several amino acids that are critical for protein synthesis, albeit at a lower level compared to glutamine. Based on these observations, they claim that the decrease in osteoblast differentiation upon asparagine depletion also occurs via a defect in protein synthesis. However, proliferation, EIF2a phosphorylation and COL1A1 levels were not affected in asparagine-deprived conditions, questioning that the decrease in differentiation is resulting from impaired protein synthesis. Further experiments to decipher the metabolic role of extracellular asparagine are therefore warranted to avoid overinterpretation of the data, including protein/matrix synthesis, analysis of amino acid levels in Asn-deprived conditions and rescue with Asn-derived metabolites.
Again, the reviewer raises a very important point. Our data indicates that Asn does contribute to amino acid biosynthesis, chiefly Asp, however, we did not evaluate the requirement of Asn for protein synthesis directly. We think it is probable that asparagine contribution to osteoblast differentiation is multifaceted. Thus, we have softened the conclusions about asparagine and the regulation of protein synthesis to reflect this uncertainty.
- To inactivate SLC1A5 in vivo, the authors use the Tet-off Osx-GFP::Cre mouse line. Importantly, newborn Osx-Cre mice display severe craniofacial abnormalities, which may complicate correct interpretation of the in vivo data, especially when analyzing at embryonic stages. Do the authors observe a similar defect in osteoblast function when SLC1A5 was deleted postnatally? This might be especially relevant because the phenotype seems to wane off over time, as knockout mice at P0 only display a craniofacial phenotype, whereas long bones appear to be normal.
The reviewer raises a very important point regarding the Sp7tTA;tetoCre line we used in this study. As mentioned, the Sp7tTA;tetoCre mice do have a partially penetrant craniofacial bone phenotype. To control for this, we only use Sp7tTA;tetoCre as “wild type” controls. In addition to the early embryonic endochondral ossification and persistent calvarial phenotypes, the Sp7tTA;tetoCre;Slc1a5^fl/fl have additional bone phenotypes compared to the Sp7tTA;tetoCre controls. This included a calvarial phenotype at both birth and 2 months of age (Figures 1 and S2). Likewise, we observe similar changes in osteoblast differentiation and bone development in the developing limbs at birth and in femurs at 2 months of age (Figure S4). Due to time constraints, we have not been able to generate sufficient numbers of mice with postnatal deletion of SLC1A5 to include here. These experiments are ongoing and will be published later.
Reviewer #3 (Public Review): This work by Sharma et al studied the role of aa transporter, ASCT2, encoded by Slc1a5 gene, that transports mostly Glmn and Asn, in osteoblasts (OB). They use gene targeting in vitro and in vivo using Sp7-Cre driven cKO. They found that ASCT2 deletion impairs OB differentiation in vitro as well as mostly intramembranous ossification in vivo by interfering with proliferation and protein synthesis. Mechanistically, they show that Glmn uptake via ASCT2 is important for aa synthesis in OBs. This group has shown before that Glmn is essential for OB metabolism. The current work further investigates this phenomenon and identifies ASCT2 as the key mechanism of Glmn uptake into OBs. The work is logically structured and carefully done with appropriate in vivo and in vitro controls. A variety of methods is used to confirm their findings, such as in vivo immunodetection and in situ hybridization and in vitro metabolic tracing. The conclusions are well justified by the data. Minor comments are: -MicroCT methodology is not adequately described and needs to be expanded
We appreciate this positive review of our work. We have modified the methods to adequately describe µCT methodology. We modified the methods as follows:
“Micro computed tomography (µCT) (VivaCT80, Scanco Medical AG) was used for three-dimensional reconstruction and analysis of bone parameters. Calvariae were harvested from either newborn mice or 2-month-old mice. All muscle and extemporaneous tissue were removed and the isolated calvariae were washed in PBS, fixed overnight in 10%NBF and dehydrated in 70% ethanol. The calvariae were immobilized in 2% agarose in PBS for scanning. A fixed volume surrounding the skull was used for 3D reconstructions. In newborn calvariae, bone volume was quantified from a fixed number of slices in the occipital lobe. The threshold was set at 280. For quantification of bone mass in the long bone, 2-month-old femurs were isolated, fixed, immobilized and scanned. Bone parameters were quantified from 200 slices directly underneath the growth plate with the threshold set at 333.”
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Evaluation Summary:
The authors report that intracellular transport of glutamine and asparagine is critical for osteoblast anabolism. The authors use a variety of in vivo and in vitro assays for the testing of their working hypothesis. The paper expands and deepens our knowledge of the role of cellular metabolism in osteoblast function and bone development.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this study, Sharma and colleagues report that intracellular transport of glutamine and asparagine in osteoblast progenitors and their descendants is critical for amino acids synthesis, overall protein synthesis, osteoblast terminal differentiation, and bone development. The authors use a variety of in vivo and in vitro assays for the testing of their working model. The paper expands and deepens our knowledge of the role of amino acid metabolism in osteoblast function and bone development.
The paper provides novel and interesting information. The authors' conclusions are largely supported by the data as shown.
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Reviewer #2 (Public Review):
Osteoblasts are highly anabolic cells that display a high proliferation rate and secrete ample amounts of extracellular matrix, indicating that these cells have a specific metabolic profile. Here, using a set of in vivo and in vitro experiments, Sharma et al describe that SLC1A5-mediated glutamine and asparagine uptake is critical to sustain osteoblast anabolism. While the experimental setup is robust, this concept has already been put forth, questioning therefore the novelty of the results. In addition, some of the author's claims are insufficiently supported by the presented data. Especially the metabolic role of asparagine in regulating osteoblast differentiation remains enigmatic. The main concerns are detailed below.
Based on their data, the authors propose that the main mechanism whereby SLC1A5 …
Reviewer #2 (Public Review):
Osteoblasts are highly anabolic cells that display a high proliferation rate and secrete ample amounts of extracellular matrix, indicating that these cells have a specific metabolic profile. Here, using a set of in vivo and in vitro experiments, Sharma et al describe that SLC1A5-mediated glutamine and asparagine uptake is critical to sustain osteoblast anabolism. While the experimental setup is robust, this concept has already been put forth, questioning therefore the novelty of the results. In addition, some of the author's claims are insufficiently supported by the presented data. Especially the metabolic role of asparagine in regulating osteoblast differentiation remains enigmatic. The main concerns are detailed below.
Based on their data, the authors propose that the main mechanism whereby SLC1A5 regulates osteoblast proliferation and differentiation is via glutamine uptake, while asparagine only contributes to a lesser extent. Importantly, the concept that glutamine metabolism regulates proliferation and differentiation of osteogenic cells by sustaining anabolic processes has already been described recently, even by the same research group (Yu Y. Cell Metab. 2019; Stegen S. JBMR 2021), questioning the novelty of the present study. Moreover, no metabolic rescue experiments were performed to unequivocally demonstrate that the defect in amino acid/protein synthesis in SLC1A5-deficient cells was causing the decrease in osteoblast proliferation and differentiation. In addition, Gln and Asn tracing (carbon and nitrogen) in SLC1A5-deficient cells would confirm that Gln and Asn uptake via SLC1A5 is important for osteoblast functioning.
Using isotopic labeling experiments, the authors demonstrate that asparagine-derived carbon and nitrogen label several amino acids that are critical for protein synthesis, albeit at a lower level compared to glutamine. Based on these observations, they claim that the decrease in osteoblast differentiation upon asparagine depletion also occurs via a defect in protein synthesis. However, proliferation, EIF2a phosphorylation and COL1A1 levels were not affected in asparagine-deprived conditions, questioning that the decrease in differentiation is resulting from impaired protein synthesis. Further experiments to decipher the metabolic role of extracellular asparagine are therefore warranted to avoid overinterpretation of the data, including protein/matrix synthesis, analysis of amino acid levels in Asn-deprived conditions and rescue with Asn-derived metabolites.
To inactivate SLC1A5 in vivo, the authors use the Tet-off Osx-GFP::Cre mouse line. Importantly, newborn Osx-Cre mice display severe craniofacial abnormalities, which may complicate correct interpretation of the in vivo data, especially when analyzing at embryonic stages. Do the authors observe a similar defect in osteoblast function when SLC1A5 was deleted postnatally? This might be especially relevant because the phenotype seems to wane off over time, as knockout mice at P0 only display a craniofacial phenotype, whereas long bones appear to be normal.
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Reviewer #3 (Public Review):
This work by Sharma et al studied the role of aa transporter, ASCT2, encoded by Slc1a5 gene, that transports mostly Glmn and Asn, in osteoblasts (OB). They use gene targeting in vitro and in vivo using Sp7-Cre driven cKO. They found that ASCT2 deletion impairs OB differentiation in vitro as well as mostly intramembranous ossification in vivo by interfering with proliferation and protein synthesis. Mechanistically, they show that Glmn uptake via ASCT2 is important for aa synthesis in OBs.
This group has shown before that Glmn is essential for OB metabolism. The current work further investigates this phenomenon and identifies ASCT2 as the key mechanism of Glmn uptake into OBs.
The work is logically structured and carefully done with appropriate in vivo and in vitro controls. A variety of methods is used to …
Reviewer #3 (Public Review):
This work by Sharma et al studied the role of aa transporter, ASCT2, encoded by Slc1a5 gene, that transports mostly Glmn and Asn, in osteoblasts (OB). They use gene targeting in vitro and in vivo using Sp7-Cre driven cKO. They found that ASCT2 deletion impairs OB differentiation in vitro as well as mostly intramembranous ossification in vivo by interfering with proliferation and protein synthesis. Mechanistically, they show that Glmn uptake via ASCT2 is important for aa synthesis in OBs.
This group has shown before that Glmn is essential for OB metabolism. The current work further investigates this phenomenon and identifies ASCT2 as the key mechanism of Glmn uptake into OBs.
The work is logically structured and carefully done with appropriate in vivo and in vitro controls. A variety of methods is used to confirm their findings, such as in vivo immunodetection and in situ hybridization and in vitro metabolic tracing. The conclusions are well justified by the data.
Minor comments are:
-MicroCT methodology is not adequately described and needs to be expanded. -
