SLC38A2 provides proline to fulfill unique synthetic demands arising during osteoblast differentiation and bone formation
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Evaluation Summary:
The paper unequivocally proves that the key function of proline during bone formation is being incorporated into proline-enriched proteins rather than contributing to other metabolic processes.
(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
Cellular differentiation is associated with the acquisition of a unique protein signature that is essential to attain the ultimate cellular function and activity of the differentiated cell. This is predicted to result in unique biosynthetic demands that arise during differentiation. Using a bioinformatic approach, we discovered that osteoblast differentiation is associated with increased demand for the amino acid proline. When compared to other differentiated cells, osteoblast-associated proteins, including RUNX2, OSX, OCN, and COL1A1, are significantly enriched in proline. Using a genetic and metabolomic approach, we demonstrate that the neutral amino acid transporter SLC38A2 acts cell-autonomously to provide proline to facilitate the efficient synthesis of proline-rich osteoblast proteins. Genetic ablation of SLC38A2 in osteoblasts limits both osteoblast differentiation and bone formation in mice. Mechanistically, proline is primarily incorporated into nascent protein with little metabolism observed. Collectively, these data highlight a requirement for proline in fulfilling the unique biosynthetic requirements that arise during osteoblast differentiation and bone formation.
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Author Response:
Reviewer #3:
Osteoblast differentiation imposes a significant metabolic demand as these cells synthesize and secrete large amounts of extracellular matrix. Recent studies have highlighted an important regulatory role for amino acid metabolism in sustaining osteoblast biosynthesis. Here, using a combinatory transcriptomic and metabolomic approach, Shen and colleagues describe that SLC38A2-dependent proline uptake is essential for osteoblast differentiation. Although the role of proline in regulating cellular properties has already been put forth in other (malignant) cells, the concept that proline contributes to specific osteoblast-related proteins is novel and interesting. However, some of the authors' claims are not sufficiently supported by the provided data and additional experiments are therefore warranted. The …
Author Response:
Reviewer #3:
Osteoblast differentiation imposes a significant metabolic demand as these cells synthesize and secrete large amounts of extracellular matrix. Recent studies have highlighted an important regulatory role for amino acid metabolism in sustaining osteoblast biosynthesis. Here, using a combinatory transcriptomic and metabolomic approach, Shen and colleagues describe that SLC38A2-dependent proline uptake is essential for osteoblast differentiation. Although the role of proline in regulating cellular properties has already been put forth in other (malignant) cells, the concept that proline contributes to specific osteoblast-related proteins is novel and interesting. However, some of the authors' claims are not sufficiently supported by the provided data and additional experiments are therefore warranted. The main concerns are detailed below.
- Based on their data, the authors state that there is a considerable enrichment of proline residues in osteoblast-related proteins (7.1%) compared to the average of all proteins (6.1%). However, it is not very clear how robust and relevant this change is, especially since other amino acids (Ala, Cys) show comparable changes. Unbiased proteomics approaches using biological replicates might therefore be warranted to avoid overinterpretation of the data.
We appreciate the reviewer’s comprehensive and thoughtful review of our study. To address the concern about cysteine, we reanalyzed our transcriptomic data to predict how cysteine demand changes during osteoblast differentiation. This analysis predicted cysteine demand declines during differentiation like alanine (data included in new Figure 1 Supplement 1C). By comparison, proline demand is predicted to increase. Consistent with these predictions, proline uptake increased significantly whereas alanine uptake was unchanged during osteoblast differentiation (See new Figure 2 Supplement 1B). These predictions led us to focus on proline specifically and is not intended to diminish the potential requirements for cysteine, alanine or other amino acids during osteoblast differentiation or bone formation. As a first step, we took a targeted approach and evaluated the effects of proline depletion on the expression of 17 distinct proteins that had various levels of proline enrichment. These data found a significant negative correlation between proline availability and protein expression based on proline composition. Based on these findings, we agree that an unbiased proteomic approach to validate the effects of proline depletion on the osteoblast proteome is warranted in future studies.
- Using 13C-proline tracing experiments, the authors show that after 72 hours more than 60% of the intracellular proline pool is 13C-labeled. They thereby claim that proline is not metabolized (line 160), although supporting data (carbon labeling of TCA cycle intermediates, glutathione, 1-Pyrroline-5-carboxylic acid) is lacking. This is especially relevant given the many metabolic fates of intracellular proline. Along the same lines, proline dehydrogenase (PRODH)-mediated proline catabolism is known to regulate electron transport chain (ETC) activity and ROS production. Are bioenergetics and/or redox homeostasis altered upon proline withdrawal or (genetic/pharmacological) SLC38A2 inactivation?
The isotopomer tracing found negligible labeling of glutamate from 13CU-proline. For this reason, we chose not to include the labeling of downstream metabolites (e.g. TCA intermediates) nor did we directly evaluate GSH which contains glutamate. We now include the data showing no labeling of the TCA intermediates malate, aKG and citrate from 13CU-proline (Figure 2 Supplement 1C). For technical reasons, we were not able to observe 1-Pyrroline-5-carboxylic acid (P5C), the product of proline oxidation by PRODH. We also did not evaluate ETC activity or ROS generation for this study. Because of the uncertainty surrounding this area we altered the discussion to address proline oxidation in the proline cycle. The relevant text is as follows:
“In addition to being directly incorporated into protein, proline can be oxidized in the inner mitochondrial membrane to form pyrroline-5-carboxylate (P5C) by proline dehydrogenase (PRODH). PRODH is a flavin dinucleotide (FAD) dependent enzyme that donates electrons to complex II of the electron transport chain coupling proline oxidation to ATP synthesis. P5C can be converted back into proline by the NADPH dependent enzyme pyrroline-5-carboxylate reductase (PYCR) in the proline cycle or can be converted into glutamate or other intermediate metabolites. Our tracing experiments did not find proline carbon enriched in either amino acids or TCA cycle intermediates. Due to technical reasons, we were not able to observe P5C in our experiments preventing us from making any conclusions about the role of proline oxidation or the contribution of proline to bioenergetics in osteoblasts. Rather, we conclude that proline is not widely metabolized past P5C in osteoblasts.”
- To study the role of SLC38A2-mediated proline uptake in bone cells in vivo, the authors use Sp7-tTA,tetO-EGFP/Cre mice. It is known that neonatal Cre-positive mice show severe craniofacial defects, which may hinder correct interpretation of the data, especially when analyzing at embryonic stages. Do the authors observe a similar phenotype in mice where SLC38A2 was deleted postnatally? The same mouse line can be used to answer this important question experimentally.
The reviewer raises a very important point regarding the Sp7-tTA;tetO-EGFP/Cre line we used in this study. As mentioned, the Sp7-tTA;tetO-EGFP/Cre mice do have a partially penetrant craniofacial bone phenotype. For this reason, we analyzed bone and molecular phenotypes in Sp7-tTA;tetO-EGFP/Cre;Slc38a2fl/fl “knockout mice” compared to Sp7-tTA;tetO-EGFP/Cre positive littermate controls. Unfortunately, we have not performed the postnatal deletions at this time. These experiments are ongoing and will be published later.
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Evaluation Summary:
The paper unequivocally proves that the key function of proline during bone formation is being incorporated into proline-enriched proteins rather than contributing to other metabolic processes.
(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):
The paper is novel and informative; the authors' conclusions are supported by the data as shown. The paper is significant as it unequivocally proves that the key function of proline during bone formation is being incorporated into proline-enriched proteins rather than contributing to other metabolic processes.
The quality of the data is outstanding. However, it would be helpful to provide better documentation of whether and how proline affects replacement of cartilage by bone during the process of endochondral bone development.
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Reviewer #2 (Public Review):
The goal of this work was to investigate the role of proline and its transporter SLC38A2 in osteoblasts (OB). OB marker proteins and regulators are enriched in proline. The authors first study the source of proline in OBs in vitro and find that it is mostly transported from outside. Then they determine that SLC38A2 amino acid transporter is a major transporter of proline in OBs. The role of proline after proline depletion and of SLC38A2 after its knockout is demonstrated by OB impairment. Genetic deletion of SLC38A2 in OB progenitors vivo, disrupts bone formation.
This is a well-designed mechanistic study done with appropriate controls and methodology. Conclusions are justified by the results. The impact for bone field is significant since OB metabolism and amino acid metabolism is not completely understood. …
Reviewer #2 (Public Review):
The goal of this work was to investigate the role of proline and its transporter SLC38A2 in osteoblasts (OB). OB marker proteins and regulators are enriched in proline. The authors first study the source of proline in OBs in vitro and find that it is mostly transported from outside. Then they determine that SLC38A2 amino acid transporter is a major transporter of proline in OBs. The role of proline after proline depletion and of SLC38A2 after its knockout is demonstrated by OB impairment. Genetic deletion of SLC38A2 in OB progenitors vivo, disrupts bone formation.
This is a well-designed mechanistic study done with appropriate controls and methodology. Conclusions are justified by the results. The impact for bone field is significant since OB metabolism and amino acid metabolism is not completely understood. Amino acid metabolism is especially important for highly synthetic cells, such as OBs.
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Reviewer #3 (Public Review):
Osteoblast differentiation imposes a significant metabolic demand as these cells synthesize and secrete large amounts of extracellular matrix. Recent studies have highlighted an important regulatory role for amino acid metabolism in sustaining osteoblast biosynthesis. Here, using a combinatory transcriptomic and metabolomic approach, Shen and colleagues describe that SLC38A2-dependent proline uptake is essential for osteoblast differentiation. Although the role of proline in regulating cellular properties has already been put forth in other (malignant) cells, the concept that proline contributes to specific osteoblast-related proteins is novel and interesting. However, some of the authors' claims are not sufficiently supported by the provided data and additional experiments are therefore warranted. The main …
Reviewer #3 (Public Review):
Osteoblast differentiation imposes a significant metabolic demand as these cells synthesize and secrete large amounts of extracellular matrix. Recent studies have highlighted an important regulatory role for amino acid metabolism in sustaining osteoblast biosynthesis. Here, using a combinatory transcriptomic and metabolomic approach, Shen and colleagues describe that SLC38A2-dependent proline uptake is essential for osteoblast differentiation. Although the role of proline in regulating cellular properties has already been put forth in other (malignant) cells, the concept that proline contributes to specific osteoblast-related proteins is novel and interesting. However, some of the authors' claims are not sufficiently supported by the provided data and additional experiments are therefore warranted. The main concerns are detailed below.
1. Based on their data, the authors state that there is a considerable enrichment of proline residues in osteoblast-related proteins (7.1%) compared to the average of all proteins (6.1%). However, it is not very clear how robust and relevant this change is, especially since other amino acids (Ala, Cys) show comparable changes. Unbiased proteomics approaches using biological replicates might therefore be warranted to avoid overinterpretation of the data.
2. Using 13C-proline tracing experiments, the authors show that after 72 hours more than 60% of the intracellular proline pool is 13C-labeled. They thereby claim that proline is not metabolized (line 160), although supporting data (carbon labeling of TCA cycle intermediates, glutathione, 1-Pyrroline-5-carboxylic acid) is lacking. This is especially relevant given the many metabolic fates of intracellular proline. Along the same lines, proline dehydrogenase (PRODH)-mediated proline catabolism is known to regulate electron transport chain (ETC) activity and ROS production. Are bioenergetics and/or redox homeostasis altered upon proline withdrawal or (genetic/pharmacological) SLC38A2 inactivation?
3. To study the role of SLC38A2-mediated proline uptake in bone cells in vivo, the authors use Sp7-tTA,tetO-EGFP/Cre mice. It is known that neonatal Cre-positive mice show severe craniofacial defects, which may hinder correct interpretation of the data, especially when analyzing at embryonic stages. Do the authors observe a similar phenotype in mice where SLC38A2 was deleted postnatally? The same mouse line can be used to answer this important question experimentally.
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