Osteocytes regulate senescence of bone and bone marrow
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eLife assessment
This paper is of potential interest for skeletal biologist studying osteocytes and skeletal aging. Using a mouse model of partial osteocyte deletion, the authors provide new understanding on the role of osteocytes in regulating other lineage cells in bone, bone marrow, and skeletal muscle. This is an important and logically presented study that offers new insight into the biology of osteocytes. The set of data from the genetic mouse model, bone phenotypic analyses, and scRNA-seq analysis largely support the conclusion.
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Abstract
The skeletal system contains a series of sophisticated cellular lineages arising from the mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) that determine the homeostasis of bone and bone marrow. Here, we reasoned that osteocyte may exert a function in regulation of these lineage cell specifications and tissue homeostasis. Using a mouse model of conditional deletion of osteocytes by the expression of diphtheria toxin subunit α in dentin matrix protein 1 (DMP1)-positive osteocytes, we demonstrated that partial ablation of DMP1-positive osteocytes caused severe sarcopenia, osteoporosis, and degenerative kyphosis, leading to shorter lifespan in these animals. Osteocytes reduction altered mesenchymal lineage commitment, resulting in impairment of osteogenesis and induction of osteoclastogensis. Single-cell RNA sequencing further revealed that hematopoietic lineage was mobilized toward myeloid lineage differentiation with expanded myeloid progenitors, neutrophils, and monocytes, while the lymphopoiesis was impaired with reduced B cells in the osteocyte ablation mice. The acquisition of a senescence-associated secretory phenotype (SASP) in both osteogenic and myeloid lineage cells was the underlying cause. Together, we showed that osteocytes play critical roles in regulation of lineage cell specifications in bone and bone marrow through mediation of senescence.
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Author Response
Reviewer #1 (Public Review):
The previous study as the authors stated showed a weaker expression of DMP1 in skeletal muscle. The authors provide a clear justification that sarcopenia-like phenotype was unlikely caused by DMP1-cre expression in muscle cells given there is no change of muscle cell numbers. It would be helpful to provide some quantification data of muscle cells to further preclude this possibility.
To define how osteocyte partial ablation was achieved, we performed the quantification of empty lacunae ratio of DTAhet mice at 13 weeks. About 80% empty lacunae was observed in DTAhet mice at 13 weeks which increased about 20% compared to 4 weeks (Line 127-131, Figure 1 – figure supplement 1B), indicating diphtheria toxin (DT) has an accumulative effect with age in DTAhet mice. We speculated that when DT …
Author Response
Reviewer #1 (Public Review):
The previous study as the authors stated showed a weaker expression of DMP1 in skeletal muscle. The authors provide a clear justification that sarcopenia-like phenotype was unlikely caused by DMP1-cre expression in muscle cells given there is no change of muscle cell numbers. It would be helpful to provide some quantification data of muscle cells to further preclude this possibility.
To define how osteocyte partial ablation was achieved, we performed the quantification of empty lacunae ratio of DTAhet mice at 13 weeks. About 80% empty lacunae was observed in DTAhet mice at 13 weeks which increased about 20% compared to 4 weeks (Line 127-131, Figure 1 – figure supplement 1B), indicating diphtheria toxin (DT) has an accumulative effect with age in DTAhet mice. We speculated that when DT accumulated to a threshold, osteocytes were ablated.
The underlying molecular mechanism is not shown in the current study, but it might be worthwhile to provide some more-depth discussions and hypotheses concerning how osteocytes could influence cell lineage commitment in bone marrow.
We thank the reviewer’s suggestion, and we now have updated this in the Discussion in the revised version (Lines 424-433).
Reviewer #3 (Public Review):
The finding that osteocyte reduction induced senescence in osteoprogenitors and myeloid lineage cells is intriguing. However, further validation of cellular senescence in bone/bone marrow is lacking. Additional approaches, such as immunostaining of key senescence markers in bone tissue sections, are needed to validate the phenotype.
According to the reviewer’s suggestion, we performed the senescence associated 𝛽galactosidase (SA-𝛽Gal) staining of frozen sections of WT and DTAhet mice femur (Figure 6 - figure supplement 1D). Accordingly, the details were given in Response to Essential Revision 2.
It is interesting that partial osteocyte ablation alters mesenchymal lineage commitment, i.e. increased adipogenesis and impaired osteogenesis. The authors should perform further analysis of their scRNA-Seq data and conduct trajectory analysis to confirm the phenomenon. Additional functional assays of bone marrow mesenchymal stem/progenitor cells, such as CFU-F and tri-lineage differentiation assays, are needed to claim the lineage commitment change of the cells.
As we used total bone marrow cells to perform scRNA-seq, the number of MSCs was not enough to perform further re-clustering and trajectory analysis. We performed GO enrichment analysis of MSC cluster which revealed that downregulated genes after osteocyte ablation were enriched in ossification and biomineral tissue development (Figure 6 - figure supplement 1E), which was consistent with the finding of impaired osteoblast differentiation (Figure 4H-J). Further, as reviewer suggested, we performed qPCR to verify related gene changes during tri-lineage differentiation. We found that the mRNA level of osteogenic markers including Alp, Ocn, Runx2 was decreased (Figure 4J), indicating the impaired osteogenesis after osteocyte ablation. Meanwhile, the mRNA level of adipogenic markers including Adipoq, Fabp4, Ppap𝛾 and Cebpa was significantly increased (Figure 6 - figure supplement 1F), indicating the promoted adipogenesis and altered MSC commitment. Besides, the mRNA level of cartilage anabolism related genes (Col1a2, Acan, Sox9 and Prg4) and catabolism related genes (Mmp3, Mmp13, Adamts1 and Adamts5) was not significantly changed (Figure 6 - figure supplement 1G), indicating that chondrogenesis was not altered after osteocyte ablation. And we now have updated this in the revised version (Lines 324-333) and trilineage differentiation methods and information of primers have been updated in Material and methods (Lines 579-590, Lines 623-637).
The mechanism why osteocyte reduction causes cellular senescence of the surrounding cells is an interesting question. It would be helpful if the authors provide evidence or give an explanation on this point. Does the phenotype recapitulate age-associated bone impairment? The laboratories of Sundeep Khosla (Mayo Clinic) and Maria Almeida (University of Arkansas for Medical Sciences) reported that osteocytes are a major cell type in bone that become senescent during aging. Although most of osteocytes were eliminated in the mouse model used in this study, were the rest osteocytes undergoing cellular senescence?
We thank the reviewer’s suggestion, and we now have updated this in the Discussion in the revised version (Line 424-433). The details were given in Response to Essential Revision 4.
We thought that the phenotypes after osteocyte ablation were similar with the ageassociated bone impairment, and to certain degree this phenotype recapitulated the ageassociated bone impairment, which further indicated the important role of osteocytes in maintaining the bone homeostasis during aging. We performed the SA-𝛽Gal staining of frozen sections of WT and DTAhet mice femur, in which we observed SA-𝛽Gal+ cell in the cortical bone region of DTAhet mice (Figure 6 - figure supplement 1D). As cortical bone mainly contains osteocyte and matrix, we inferred that the rest osteocytes may also underwent cellular senescence.
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eLife assessment
This paper is of potential interest for skeletal biologist studying osteocytes and skeletal aging. Using a mouse model of partial osteocyte deletion, the authors provide new understanding on the role of osteocytes in regulating other lineage cells in bone, bone marrow, and skeletal muscle. This is an important and logically presented study that offers new insight into the biology of osteocytes. The set of data from the genetic mouse model, bone phenotypic analyses, and scRNA-seq analysis largely support the conclusion.
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Reviewer #1 (Public Review):
The study by Ding et al. reports a new mechanism concerning the deletion of bone osteocytes and changes of MSC and HSC linkages in bone marrow. A heterozygous mouse model by DMP-1-driven DTA expression was used to remove osteocytes given that homozygous mice are embryonic lethal. The authors showed that partial deletion cause severe bone loss in both trabecular and cortical bone as well as sarcopenia, osteoporosis and degenerative kyphosis. With single cell RNA seq, they found that osteocyte deletion affects both MSC and HSC lineage commitment. Consistent with bone phenotypes, MSC showed reduced osteogenic differentiation. scRNA seq of HSC showed an increase of myeloid progenitors, neutrophils and monocytes, but a decrease of lymphopoiesis. Interestingly, multiple senescence genes are upregulated, implying …
Reviewer #1 (Public Review):
The study by Ding et al. reports a new mechanism concerning the deletion of bone osteocytes and changes of MSC and HSC linkages in bone marrow. A heterozygous mouse model by DMP-1-driven DTA expression was used to remove osteocytes given that homozygous mice are embryonic lethal. The authors showed that partial deletion cause severe bone loss in both trabecular and cortical bone as well as sarcopenia, osteoporosis and degenerative kyphosis. With single cell RNA seq, they found that osteocyte deletion affects both MSC and HSC lineage commitment. Consistent with bone phenotypes, MSC showed reduced osteogenic differentiation. scRNA seq of HSC showed an increase of myeloid progenitors, neutrophils and monocytes, but a decrease of lymphopoiesis. Interestingly, multiple senescence genes are upregulated, implying premature aging in bone marrow.
Strengths:
The paper shows a new mechanism that numbers of healthy osteocytes have a major impact on differentiation and commitment of bone marrow MSC and HSC progenitor cells and bone marrow cell senescence.
Various thorough techniques (microCT, bone formation and bone histomorphometry, histochemistry) were used to study the bone properties. The data convincing show that partial ablation of osteocytes leads to severe bone loss and compromised bone structure. Interestingly, it does not just affect bone, but has a major effect on muscle. The consist phenotypes are manifested in both male and female mice.
In addition to expected MSC data, they further conducted scRNA seq with HSC and showed the increased cell population in myeloid, monocyte linkage, but reduce in lymphogenic progenitors. Further analysis revealed increased gene expression related to senescence-associated secretory phenotype (SASP). All evidence including reduced viable osteocytes, reduced bone mass and osteogenic commitment of MSC, and increased myeloid and senescence is related to premature aging.
The experiments in general were well designed and conducted with compressive characterization, and the data supported the conclusions. The paper is also logically written and figures were well presented providing clear graphic illustrations.Minor weaknesses:
The previous study as the authors stated showed a weaker expression of DMP1 in skeletal muscle. The authors provide a clear justification that sarcopenia-like phenotype was unlikely caused by DMP1-cre expression in muscle cells given there is no change of muscle cell numbers. It would be helpful to provide some quantification data of muscle cells to further preclude this possibility.
The underlying molecular mechanism is not shown in the current study, but it might be worthwhile to provide some more-depth discussions and hypotheses concerning how osteocytes could influence cell lineage commitment in bone marrow. -
Reviewer #2 (Public Review):
This manuscript addresses the function of osteocytes that are not well understood. These cells are embedded in the largest organ, bone. In addition to mechanosensing, the concept that bone, and that of the osteocytes can act as endocrine cells, communicating with other organs with soluble factors is beginning to take shape. In addressing the function of osteocytes in mice, the authors specially remove/reduce the number of osteocytes using genetic tools to conditionally activate the expression of diphtheria toxin (DTA) in osteocytes that are expressing the DMP1, thus, killing these cells. The impact on the skeletal system in development and ageing were studied, as well as cells in the bone marrow.
Mice with completed removal of DMP1-expressing osteocytes die before birth. However, mice with partially reduced …
Reviewer #2 (Public Review):
This manuscript addresses the function of osteocytes that are not well understood. These cells are embedded in the largest organ, bone. In addition to mechanosensing, the concept that bone, and that of the osteocytes can act as endocrine cells, communicating with other organs with soluble factors is beginning to take shape. In addressing the function of osteocytes in mice, the authors specially remove/reduce the number of osteocytes using genetic tools to conditionally activate the expression of diphtheria toxin (DTA) in osteocytes that are expressing the DMP1, thus, killing these cells. The impact on the skeletal system in development and ageing were studied, as well as cells in the bone marrow.
Mice with completed removal of DMP1-expressing osteocytes die before birth. However, mice with partially reduced osteocytes survive with reduced life span associated with severe osteoporosis, kyphosis and sarcopenia, conditions that are age-related, and the authors claimed an association with prematured ageing. The authors showed changes in the balance between the osteoblast, osteoclast and adipocyte lineages as possible mechanisms.
A relationship between bone and muscle is known, especially the contractile muscles. Their finding that there is a continuing body and muscle weight lost substantiates this relationship with focal muscle atrophy and sarcopenia.
Using a similar genetic approach, a previous study by Asada et al (2013) has shown that osteocytes regulate mobilization of haematopoietic stem/progenitor cells in mice. This manuscript extends this relationship in an assessment of the bone marrow cells using single cell RNA sequencing (scRNA-seq), revealing an alteration of the haematopoietic lineage commitment, with a shift from lymphopoiesis to myelopoiesis.
The most novel and interesting finding is the association with senescence. However, this is also perhaps the weakest link in the manuscript, as it presents a big jump in the hypothesis from the single cell data. The hypothesis was substantiated from an assessment of a senescence associated secretory phenotype (ASAP) score from the scRNA-seq data, which was not well explained. Nevertheless, circulatory SASP were elevated in osteocyte compromised mice, and concluded that osteocyte reduction induced senescence in osteoprogenitors and myeloid lineage cells.
Overall, the manuscript was logically presented, and the data in most parts supported the conclusion. The relationship however was mostly through descriptive morphological and biochemical analyses of the mutant mice. While there are weaker areas that need to be further strengthened, there are novel findings providing further insights into the biology of osteocytes and reaffirms the concept of bone as an endocrine organ.
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Reviewer #3 (Public Review):
The authors studied the impact of partial ablation of osteocytes on the changes of musculoskeletal system. Using a mouse model of partial osteocyte deletion by the expression of DTA in DMP-1-positive osteocytes (DTRhet), the authors demonstrated an interesting phenotype with multi-organ deficits. Particularly, the authors found that DTRhet mice have severe osteoporosis, kyphosis, sarcopenia with shorter life span. By assessing the cellular changes in bone/bone marrow, the authors showed that partial osteocyte ablation increased adipogenesis, impaired osteogenesis and promoted osteoclastogenesis. They went on to show that osteocyte ablation altered hematopoietic lineage, characterized by the shift from lymphopoiesis to myelopoiesis. Finally, they conducted scRNA-seq and found that total bone marrow from …
Reviewer #3 (Public Review):
The authors studied the impact of partial ablation of osteocytes on the changes of musculoskeletal system. Using a mouse model of partial osteocyte deletion by the expression of DTA in DMP-1-positive osteocytes (DTRhet), the authors demonstrated an interesting phenotype with multi-organ deficits. Particularly, the authors found that DTRhet mice have severe osteoporosis, kyphosis, sarcopenia with shorter life span. By assessing the cellular changes in bone/bone marrow, the authors showed that partial osteocyte ablation increased adipogenesis, impaired osteogenesis and promoted osteoclastogenesis. They went on to show that osteocyte ablation altered hematopoietic lineage, characterized by the shift from lymphopoiesis to myelopoiesis. Finally, they conducted scRNA-seq and found that total bone marrow from DTRhet mice (vs. WT mice) had increased senescence featured by higher SASP score. The authors reach the major conclusion that osteocytes play critical roles in regulating lineage cell specifications in bone and bone marrow by inducing organismal senescence. This is a very interesting set of studies, in which most of the authors' conclusions are supported by well-established mouse genetic conditional approaches and skeletal phenotypic analyses.
I have the following points for the authors to address:
1. The finding that osteocyte reduction induced senescence in osteoprogenitors and myeloid lineage cells is intriguing. However, further validation of cellular senescence in bone/bone marrow is lacking. Additional approaches, such as immunostaining of key senescence markers in bone tissue sections, are needed to validate the phenotype.
2. It is interesting that partial osteocyte ablation alters mesenchymal lineage commitment, i.e. increased adipogenesis and impaired osteogenesis. The authors should perform further analysis of their scRNA-Seq data and conduct trajectory analysis to confirm the phenomenon. Additional functional assays of bone marrow mesenchymal stem/progenitor cells, such as CFU-F and tri-lineage differentiation assays, are needed to claim the lineage commitment change of the cells.
3. The mechanism why osteocyte reduction causes cellular senescence of the surrounding cells is an interesting question. It would be helpful if the authors provide evidence or give an explanation on this point. Does the phenotype recapitulate age-associated bone impairment? The laboratories of Sundeep Khosla (Mayo Clinic) and Maria Almeida (University of Arkansas for Medical Sciences) reported that osteocytes are a major cell type in bone that become senescent during aging. Although most of osteocytes were eliminated in the mouse model used in this study, were the rest osteocytes undergoing cellular senescence? -
