Bones have diverse roles in the body, such as supporting weight, allowing movement and protecting internal organs. Regardless of their location, all bones in the body are formed and maintained by specialized cells called osteoblasts. To produce the different components of bone, osteoblasts need a constant supply of amino acids, the building blocks of proteins. If these nutrients are limited, this may lead to weak and fragile bones that can fracture more easily.

Naïve cells in the bone, which are yet to have a defined role, also require large amounts of amino acids to develop into fully functioning osteoblasts. Previous studies have found that specific amino acids (like glutamine and asparagine) are particularly important for forming the proteins in bone. However, it was unclear which amino acids are critical for the development of osteoblasts.

To investigate, Shen et al. studied naïve cells that had been extracted from the embryos of mice and developed into osteoblasts in the laboratory. They found that the developing osteoblasts produced proteins enriched in proline, and that naïve cells required large amounts of this amino acid as they turned into osteoblasts.

Genetic analysis revealed that osteoblasts carry the gene for a protein called SLC38A2, which has been shown to transport proline into other types of cells. Shen et al. then used gene editing tools to delete this transporter from the osteoblasts of mice. The mutated mice could not efficiently produce proline-rich proteins during embryonic development and formed less bone.

These findings highlight that proline is important for developing osteoblasts and synthesizing the products of bone. Further research is needed, but it is possible that dietary supplements of proline may be beneficial for maintaining or promoting bone formation in adulthood. This could help individuals that have more fragile bones, such as the elderly or patients with bone diseases, like osteoporosis.

The mammalian boney skeleton is a remarkable organ that has multiple functions, including support, mobility, protection of internal organs, endocrine signaling, mineral storage, as well as being a site for red blood cell production (Guntur and Rosen, 2012; Jagannathan-Bogdan and Zon, 2013; Long, 2011; Salhotra et al., 2020). The skeleton develops embryonically through two distinct mechanisms, intramembranous and endochondral ossification (Berendsen and Olsen, 2015). Intramembranous ossification is responsible for forming the ‘flat’ bones of the skull. Here, mesenchymal progenitor cells condense and give rise to bone directly. The remainder of the skeleton develops through endochondral ossification. In this process, the mesenchymal progenitors condense and give rise to a cartilaginous template that is subsequently ossified. Regardless of the developmental mechanism, skeletal development depends upon osteoblasts. Osteoblasts are secretory cells responsible for producing and secreting the collagen type 1 (COL1A1)-rich extracellular bone matrix. Osteoblast differentiation is tightly regulated by the transcription factors RUNX2 and OSX (encoded by Sp7) (Ducy et al., 1997; Nakashima et al., 2002; Otto et al., 1997; Takarada et al., 2016). Genetic studies in mice demonstrate that RUNX2 is essential for commitment to the osteoblast lineage as well as the transcriptional regulation of osteoblast marker genes (e.g., Spp1 and Bglap) (Komori et al., 1997; Meyer et al., 2014; Otto et al., 1997; Wu et al., 2014). OSX functions downstream of RUNX2 to regulate osteoblast differentiation and osteoblast gene expression (e.g., Spp1, Ibsp, and Bglap) (Bianco et al., 1991; Ducy et al., 1996).

During differentiation, osteoblasts acquire a distinct protein profile in addition to increasing bone matrix production (Alves et al., 2010; Zhang et al., 2007). Protein and bone matrix production is biosynthetically demanding and predicted to present differentiating osteoblasts with changing metabolic demands (Buttgereit and Brand, 1995). Thus, osteoblasts must maximize nutrient and amino acid acquisition for differentiation and matrix production to proceed. Consistent with this, both glucose and amino acid uptake are required for osteoblast differentiation and bone formation (Elefteriou et al., 2006; Rached et al., 2010; Wei et al., 2015). Osteoblasts primarily rely on glycolytic metabolism of glucose that provides ATP for protein synthesis and to regulate RUNX2 stability to promote osteoblast differentiation (Esen et al., 2013; Lee et al., 2020; Wei et al., 2015). Like glucose, amino acids have long been recognized as important regulators of osteoblast differentiation and bone matrix production (Elefteriou et al., 2006; Hahn et al., 1971; Karner et al., 2015; Rached et al., 2010; Shen and Karner, 2021; Yu et al., 2019). Affecting the ability of cells to sense or obtain amino acids either by limiting their availability in the media or inhibiting cellular uptake has detrimental effects on osteoblast differentiation and bone formation (Chen and Long, 2018; Elefteriou et al., 2006; Esen et al., 2013; Hu et al., 2020; Karner et al., 2015; Rached et al., 2010; Shen et al., 2021; Yu et al., 2019). Despite this, the role of individual amino acids in osteoblasts is not well understood. Recent studies identified glutamine as a particularly important amino acid in osteoblasts supporting protein and amino acid synthesis, redox regulation, and energetics (Karner et al., 2015; Shen et al., 2021; Stegen et al., 2021; Yu et al., 2019). Whether other individual amino acids are similarly important for osteoblast differentiation remains unknown.

Proline is an intriguing amino acid in osteoblasts as it is important for both the biosynthesis and structure of collagen (Grant and Prockop, 1972; Krane, 2008). In addition, interest in proline has recently increased as proline is critical for cancer cell survival, tumorigenesis, and metastasis (Elia et al., 2017; Liu et al., 2012a; Nagano et al., 2017; Olivares et al., 2017; Phang, Liu, Hancock, & Christian, 2012). Proline is a multifunctional amino acid with important roles in carbon and nitrogen metabolism, oxidative stress protection, cell signaling, nutrient adaptation, and cell survival (Hollinshead et al., 2018; Liu et al., 2012c; Phang, 2019). Proline can contribute to protein synthesis directly through incorporation into protein or can be metabolized into downstream products involved in energetic and biosynthetic reactions. Despite its emerging role in cancer cells, the role of proline during osteoblast differentiation and bone development is understudied.

Here, we identify proline as a critical nutrient in osteoblasts. Using a multifaceted approach, we demonstrate that sodium-dependent neutral amino acid transporter-2 (SNAT2, encoded by Slc38a2 and denoted herein as SLC38A2) acts cell-autonomously to provide proline necessary for osteoblast differentiation and bone development. Mechanistically, proline is essential for the synthesis of proline-rich osteoblast proteins, including those that regulate osteoblast differentiation (e.g., RUNX2 and OSX) and bone matrix production (e.g., COL1A1). These data highlight a broad requirement for proline to fulfill unique synthetic demands associated with osteoblast differentiation and bone formation.

Here, we have defined a specific requirement for proline that arises during osteoblast differentiation and bone formation. Namely, that osteoblasts require proline to fulfill unique biosynthetic demands that arise due to increased production of proline-enriched osteoblast-associated proteins. To fulfill this demand, osteoblasts significantly increase proline consumption during differentiation. Genetically limiting proline uptake by ablating the proline transporter SLC38A2 results in delayed bone development. Mechanistically, osteoblasts utilize proline primarily for the synthesis of proline-enriched osteoblast proteins to facilitate both osteoblast differentiation and bone matrix production. Collectively, these data highlight a broad requirement for proline to regulate osteoblast differentiation and bone development in addition to supporting collagen synthesis.

Osteoblast differentiation is characterized by a distinct protein profile in addition to increasing bone matrix production (Alves et al., 2010; Zhang et al., 2007). These osteoblast-associated proteins are enriched for the amino acid proline compared to all other proteins (Figure 1, Tables 1–2). We and others have recently described increased consumption of numerous amino acids in differentiating osteoblasts including glutamine (Sharma et al., 2021; Stegen et al., 2021; Yu et al., 2019), asparagine (Sharma et al., 2021), and proline (this study). Glutamine and asparagine contribute to both de novo amino acid biosynthesis and protein synthesis directly (Sharma et al., 2021). 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 (Elia et al., 2017; Liu and Phang, 2012b; Olivares et al., 2017; Phang et al., 2012). 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 trichloroacetic acid (TCA) intermediates. Due to technical reasons, we were not able to observe P5C in our experiments. For this reason, we conclude that proline is not widely metabolized past P5C in osteoblasts, but we are unable to make any conclusions about proline oxidation or the contribution of proline to bioenergetics in osteoblasts. Rather, our data indicates the primary use for proline is direct incorporation into nascent protein. Consistent with this, reducing proline availability specifically reduced the synthesis of proteins with higher-than-average proline content without affecting mTOR activation or inducing ISR (Figures 3 and 4). Proline depletion likely results in stalling of proline codons of proline-enriched genes at both the A and P sites of the ribosome similar to clear cell renal cell carcinoma that are vulnerable to proline depletion (Loayza-Puch et al., 2016). Consistent with this, protein expression was negatively correlated with the proline content in proteins when proline availability or uptake was limited (Figures 3 and 4). By comparison, limiting the availability of glutamine-induced robust activation of the ISR inhibits global protein synthesis (Sharma et al., 2021). This likely reflects the necessity of glutamine metabolism to maintain amino acid concentrations (including proline) and provide other metabolites during osteoblast differentiation (Sharma et al., 2021; Stegen et al., 2021; Yu et al., 2019). Consistent with the more direct use of proline in protein but not amino acid biosynthesis, we did not observe activation of the ISR in proline-free conditions despite reduced charging of proline tRNA. It is important to note that we evaluated the effects of proline withdrawal for 48 hr. This time point may miss the chronic effects of proline withdrawal as proline uptake is slow and the intracellular proline pool is stable with low turnover in naïve calvarial cells (Figure 2). Under these conditions, de novo biosynthesis of proline may be sufficient to meet the basal needs of naïve calvarial cells. Regardless, proline removal results in reduced synthetic efficiency of proline-rich proteins. This effect is likely exacerbated during osteoblast differentiation as these proline-rich proteins are increased.

Previous studies characterized proline uptake in both bones and osteoblasts directly. These studies described proline uptake occurring primarily via System A but did not identify individual transporters mediating proline uptake (Adamson and Ingbar, 1967; Finerman and Rosenberg, 1966; Hahn et al., 1969; Yee, 1988). Here, we identified the sodium-dependent neutral amino acid transporter SLC38A2 as responsible for approximately 55% of proline uptake in both calvarial osteoblasts and isolated bones (Figure 4B, Figure 5—figure supplement 3B). This is consistent with previous reports that System A mediates 60% of proline uptake in osteoblasts (Yee, 1988). Interestingly, SLC38A2 ablation affected only proline uptake (Figure 5—figure supplement 3B). It is not clear why SLC38A2 exclusively transports proline in osteoblasts as amino acid transporters are thought to be promiscuous in their substrate specificity (Kandasamy et al., 2018; Teichmann et al., 2017). For example, SLC38A2 is reported to transport alanine, serine, glycine, and glutamine in different cellular contexts (Bröer et al., 2016; Morotti et al., 2019). Our data indicates that glutamine is not a primary substrate for SLC38A2 in bone cells. This is consistent with our recent data demonstrating that glutamine uptake is mediated primarily by System ASC with no involvement of System A in osteoblasts (Sharma et al., 2021; Shen et al., 2021). In light of these data, a better understanding of the molecular regulation of SLC38A2 activity and substrate specificity is needed. In addition, it will be important to identify the transporters mediating SLC38A2-independent proline uptake as well as to understand their function during osteoblast differentiation and bone development.

Reducing proline uptake inhibited bone development in mice (Figure 5). This phenotype was attributed primarily to decreased osteoblast differentiation and reduced bone matrix production. Osteoblast differentiation and bone matrix production are associated with a unique biosynthetic demand for proline. Using a bioinformatic approach, we discovered that osteoblast-associated proteins are more enriched for proline than any other amino acid when compared to other cell types (Figure 1). Many of these proline-rich proteins are essential regulators of osteoblast differentiation (e.g., RUNX2, OSX, and ATF4), bone matrix production (e.g., COL1A1), or regulate the endocrine functions of bone (e.g., OCN) (Ducy et al., 1996; Ducy et al., 1997; Elefteriou et al., 2006; Kern et al., 2001; Nakashima et al., 2002; Otto et al., 1997; Yang et al., 2004). Limiting proline availability by genetically ablating SLC38A2 specifically affected the production of proline-rich proteins (e.g., RUNX2, OSX, and COL1A1) in a manner that was proportional to the relative proline content. It is important to note that relatively minor reductions in protein expression or function are known to negatively impact osteoblast differentiation and bone development and underlie human bone diseases (Baek et al., 2013; Bardai et al., 2016; Ben Amor et al., 2013; Choi et al., 2001; Lapunzina et al., 2010; Lee et al., 1997; Lou et al., 2009; Mundlos et al., 1997; Zhang et al., 2009). Thus, ablating SLC38A2-dependent proline uptake has broad effects on osteoblast differentiation due to minor reductions in many proline-rich osteoblast regulatory proteins. This highlights an unappreciated mechanism by which osteoblast differentiation is vulnerable to nutrient (e.g., proline) limitations. When proline is available, osteoblast progenitors efficiently synthesize the proline-rich proteins necessary for differentiation (RUNX2 and OSX) and bone matrix deposition (COL1A1). The high proline content of these proteins presents a novel cellular checkpoint to ascertain if appropriate resources, in this case proline, are available for osteoblast differentiation to proceed. When proline is limited, these proteins are not efficiently synthesized, which limits osteoblast differentiation and bone matrix production until sufficient proline is available. This is critical to ensure that cells can meet the synthetic challenges associated with osteoblast differentiation and bone matrix production.

In addition to the aforementioned osteoblast defects, we also observed delayed COLX remodeling in Sp7Cre;Slc38a2^fl/fl mice. This suggests that loss of SLC38A2 in hypertrophic chondrocytes contributes to delayed endochondral ossification in Sp7Cre;Slc38a2^fl/fl mice. At this time, it is not apparent how SLC38A2 affects either chondrocyte differentiation or hypertrophy. It is intriguing to speculate that SLC38A2 provides chondrocytes with proline (or other amino acids) to facilitate the production of proline-rich collagens (e.g., COL2 and COLX) or other proteins during chondrogenesis and endochondral ossification. While the role of SLC38A2 in chondrogenesis was not a focus of this work, it will be important to determine both the necessity and the molecular substrates of SLC38A2 during chondrogenesis.

In summary, we have defined the necessity and the molecular substrates of Slc38a2 in osteoblasts. Our data indicate that SLC38A2 acts cell-autonomously in osteoblasts to provide proline and that SLC38A2 is the major proline transporter in osteoblasts. Proline is essential for the production of proline-rich transcription factors (e.g., RUNX2 and OSX) and matrix proteins (COL1A1) necessary for osteoblast differentiation and bone formation. These data expand our understanding of the regulation of proline uptake and usage in osteoblasts and underscore the necessity of proline for osteoblast differentiation and bone development.

All data generated or analyzed during this study are included in this submission and the supporting files. Source data files are included for all western blot images and excel spreadsheets are included for the RNAseq and metabolic tracing experiments in figures 1 and 2.

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Author details

1. Leyao Shen

   1. Department of Orthopaedic Surgery, Duke University School of Medicine, Durham, United States
   2. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Investigation, Writing - original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4952-437X

2. Yilin Yu

   Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Yunji Zhou

   Department of Biostatistics and Bioinformatics, Duke University School of Medicine, Durham, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

4. Shondra M Pruett-Miller

   Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3793-585X

5. Guo-Fang Zhang

   1. Sarah W. Stedman Nutrition and Metabolism Center & Duke Molecular Physiology Institute, Duke University Medical Center, Durham, United States
   2. Department of Medicine, Duke University School of Medicine, Durham, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

6. Courtney M Karner

   1. Department of Orthopaedic Surgery, Duke University School of Medicine, Durham, United States
   2. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States
   3. Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center at Dallas, Dallas, United States

   Contribution

   Conceptualization, Investigation, Supervision, Writing - review and editing

   For correspondence

   courtney.karner@utsouthwestern.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0387-4486

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