O-GlcNAc glycosylation orchestrates fate decision and niche function of bone marrow stromal progenitors
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eLife assessment:
Bone marrow stromal cells (BMSCs) can differentiate into a variety of cell types such as osteoblasts, chondrocytes, and adipocytes. The authors of this important study provide compelling and strong evidence that ablating O-GlcNAc transferase (OGT) in BMSCs impairs bone formation but promotes marrow adiposity. The results show that the balance of osteogenic and adipogenic differentiation of BMSCs is controlled by reciprocal O-GlcNAc regulation of lineage-specifying transcription factors, and highlights the importance of an intracellular glycosylation process of specific proteins in bone formation and bone marrow adipocytes.
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Abstract
In mammals, interactions between the bone marrow (BM) stroma and hematopoietic progenitors contribute to bone-BM homeostasis. Perinatal bone growth and ossification provide a microenvironment for the transition to definitive hematopoiesis; however, mechanisms and interactions orchestrating the development of skeletal and hematopoietic systems remain largely unknown. Here, we establish intracellular O-linked β-N-acetylglucosamine (O-GlcNAc) modification as a posttranslational switch that dictates the differentiation fate and niche function of early BM stromal cells (BMSCs). By modifying and activating RUNX2, O-GlcNAcylation promotes osteogenic differentiation of BMSCs and stromal IL-7 expression to support lymphopoiesis. In contrast, C/EBPβ-dependent marrow adipogenesis and expression of myelopoietic stem cell factor (SCF) is inhibited by O-GlcNAcylation. Ablating O-GlcNAc transferase (OGT) in BMSCs leads to impaired bone formation, increased marrow adiposity, as well as defective B-cell lymphopoiesis and myeloid overproduction in mice. Thus, the balance of osteogenic and adipogenic differentiation of BMSCs is determined by reciprocal O-GlcNAc regulation of transcription factors, which simultaneously shapes the hematopoietic niche.
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Author Response
Reviewer #2 (Public Review):
Here I will mainly comment on the biology of adipocytes, which is my specialty.
In this manuscript, it has been very convincingly shown that O-GlcNAc acts as an important regulator of MSC differentiation in mice, and given previous studies in which O-GlcNAc is regulated by aging and nutritional status, it makes sense that this PTM determines differentiation and BM niche.
The point that O-GlcNAc regulates adipocyte differentiation is convincing, but there are already previous studies using 3T3-L1 (e.g., Biochemical and Biophysical Research Communications 417 (2012) 1158-1163), and a more step-by-step demonstration of the molecular mechanism would make this an excellent paper that can be extended to adipocyte research in general, not just BM.
While O-GlcNAc has been demonstrated in …
Author Response
Reviewer #2 (Public Review):
Here I will mainly comment on the biology of adipocytes, which is my specialty.
In this manuscript, it has been very convincingly shown that O-GlcNAc acts as an important regulator of MSC differentiation in mice, and given previous studies in which O-GlcNAc is regulated by aging and nutritional status, it makes sense that this PTM determines differentiation and BM niche.
The point that O-GlcNAc regulates adipocyte differentiation is convincing, but there are already previous studies using 3T3-L1 (e.g., Biochemical and Biophysical Research Communications 417 (2012) 1158-1163), and a more step-by-step demonstration of the molecular mechanism would make this an excellent paper that can be extended to adipocyte research in general, not just BM.
While O-GlcNAc has been demonstrated in regulating many aspects of metabolic physiology, our understanding of its role in adipogenesis has been limited so far. As the reviewer pointed out, there was an in vitro report on its inhibition of adipogenesis in 3T3-L1 cells (Ji et al., 2012). Two recent publications from Dr. Xiaoyong Yang’s group revealed the profound role of mature white adipocytes OGT in regulating lipolysis and obesity (Li et al., 2018; Yang et al., 2020). To my knowledge, our manuscript is the first attempt to address the regulation of adipogenesis by O-GlcNAc in vivo. While using the BMSCs as a non-conventional model, we speculate our molecular mechanisms (i.e., O-GlcNAc inhibition of C/EBPβ) could be conserved in peripheral adipose organs, including white and brown adipose tissues. Future experiments are warranted in the lab to extend the current knowledge to these adipocyte progenitors. Nonetheless, I would also like to point out that, due to the broad actions of OGT and the current lack of adipocyte progenitor specific Cre animal tools, such efforts might be futile as results can be confounded by defects in other organs/cells.
It is somewhat unclear whether or not the authors' in vitro experiments using 10T1/2 cells accurately reflect what is happening in vivo in knockout mice. The PDGFRa+VCAM1+ population of adipocyte progenitors shown by the authors is upregulated by about 30% by knockout of Ogt (Figure 4C). How significant is this difference? Rather, might the expression of Pparg, which indicates lineage commitment, be the underlying mechanism? In any case, this manuscript is highly impactful in the sense that the differentiation of adipocytes forming the BM niche can be controlled using tissue-specific knockouts of the Ogt gene.
We agree with the reviewer that the role of OGT in BMSC fate determination and adipogenesis might be multifaceted. The 30% increase in PDGFRa+VCAM1+ BM adipose progenitors cannot fully explain the massive adipogenesis observed in OgtΔOsx animals (Fig. 4A). Indeed, we provided in vitro evidence that genetic deletion or chemical inhibition of OGT activates adipogenesis (Fig. 4D-I). Mechanistically, we found the O-GlcNAcylation of C/EBPβ protein (but not PPARγ) is responsible in the inhibition, which leads to reduced expression of adipogenic genes, including Pparg (Fig. 4H).
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eLife assessment:
Bone marrow stromal cells (BMSCs) can differentiate into a variety of cell types such as osteoblasts, chondrocytes, and adipocytes. The authors of this important study provide compelling and strong evidence that ablating O-GlcNAc transferase (OGT) in BMSCs impairs bone formation but promotes marrow adiposity. The results show that the balance of osteogenic and adipogenic differentiation of BMSCs is controlled by reciprocal O-GlcNAc regulation of lineage-specifying transcription factors, and highlights the importance of an intracellular glycosylation process of specific proteins in bone formation and bone marrow adipocytes.
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Reviewer #1 (Public Review):
This paper identifies an intracellular O-GlcNAc glycosylation of specific proteins in the control of bone formation and bone marrow adiposity. Compelling evidence is provided for the role of OGT-mediated O-GlcNAc glycosylation of RUNX2 in osteogenic differentiation versus OGT-mediated O-GlcNAc glycosylation of C/EBPβ in bone marrow adipogenesis.
Overall, the experiments have been done with great rigor, and sufficient details are provided for reproducibility. The authors developed a novel concept in the control of bone formation and bone marrow adiposity.
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Reviewer #2 (Public Review):
Here I will mainly comment on the biology of adipocytes, which is my specialty.
In this manuscript, it has been very convincingly shown that O-GlcNAc acts as an important regulator of MSC differentiation in mice, and given previous studies in which O-GlcNAc is regulated by aging and nutritional status, it makes sense that this PTM determines differentiation and BM niche.
The point that O-GlcNAc regulates adipocyte differentiation is convincing, but there are already previous studies using 3T3-L1 (e.g., Biochemical and Biophysical Research Communications 417 (2012) 1158-1163), and a more step-by-step demonstration of the molecular mechanism would make this an excellent paper that can be extended to adipocyte research in general, not just BM.
It is somewhat unclear whether or not the authors' in vitro …
Reviewer #2 (Public Review):
Here I will mainly comment on the biology of adipocytes, which is my specialty.
In this manuscript, it has been very convincingly shown that O-GlcNAc acts as an important regulator of MSC differentiation in mice, and given previous studies in which O-GlcNAc is regulated by aging and nutritional status, it makes sense that this PTM determines differentiation and BM niche.
The point that O-GlcNAc regulates adipocyte differentiation is convincing, but there are already previous studies using 3T3-L1 (e.g., Biochemical and Biophysical Research Communications 417 (2012) 1158-1163), and a more step-by-step demonstration of the molecular mechanism would make this an excellent paper that can be extended to adipocyte research in general, not just BM.
It is somewhat unclear whether or not the authors' in vitro experiments using 10T1/2 cells accurately reflect what is happening in vivo in knockout mice. The PDGFRa+VCAM1+ population of adipocyte progenitors shown by the authors is upregulated by about 30% by knockout of Ogt (Figure 4C). How significant is this difference? Rather, might the expression of Pparg, which indicates lineage commitment, be the underlying mechanism? In any case, this manuscript is highly impactful in the sense that the differentiation of adipocytes forming the BM niche can be controlled using tissue-specific knockouts of the Ogt gene.
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Reviewer #3 (Public Review):
This study has the strengths of novelty and significance across multiple fields, including bone marrow biology, skeletal health, hematopoiesis, and protein posttranslational modification (PTM). It establishes the role of protein O-GlcNAcylation in bone development and bone marrow niche. The cooperative O-GlcNAcylation on Runx2 and C/EBPb to prime BMSCs toward osteoblast differentiation over adipogenesis is a very interesting and sounding molecular mechanism. The employment of an inducible OGT conditional knockout mouse model with appropriate Osx-Cre controls is conclusive and rigorous. The in vitro experiments were carefully designed in support of strong rationales. The overall flow of the story is logical and clear. Last, the conclusions are drawn from concrete evidence in an accurate way.
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