Impaired ERK MAPK activation in mature osteoblasts enhances bone formation via the mTOR pathway
Abstract
Emerging evidence supports that osteogenic differentiation of skeletal stem cells (SSCs) is a key determinant of overall bone formation and bone mass. Despite extensive studies showing mitogen-activated protein kinase (MAPK) function in osteoblast differentiation, none of these studies properly show in vivo evidence of impacting post-lineage commitment and subsequent maturation. Here, we describe how the extracellular signal-regulated kinase (ERK) pathway in osteoblasts controls bone formation by suppressing the mechanistic target of rapamycin (mTOR) pathway. We also show that, while ERK inhibition blocks the differentiation of osteogenic precursors when initiated at an early stage, ERK inhibition surprisingly promotes the later stages of osteoblast differentiation. Accordingly, inhibition of the ERK pathway using a small compound inhibitor or conditional deletion of the MAP2Ks Mek1 and Mek2 , in mature osteoblasts and osteocytes ( Mek1/2 Dmp1 ), markedly increased bone formation due to augmented osteoblast differentiation. Mice with inducible deletion of the ERK pathway in mature osteoblasts ( Mek1/2 Ocn-Ert ) also displayed similar phenotypes, demonstrating that this phenotype reflects continuous postnatal inhibition of late-stage osteoblast maturation. Mechanistically, ERK inhibition increases mitochondrial function and SGK1 phosphorylation via mTOR2 activation, which leads to osteoblast differentiation and production of angiogenic and osteogenic factors to promote bone formation. This phenotype was partly reversed by inhibiting mTOR. Our study uncovers a surprising dichotomy of ERK pathway functions in osteoblasts, whereby ERK activation promotes the early differentiation of osteoblast precursors, but inhibits the subsequent differentiation of committed osteoblasts via mTOR-mediated regulation of mitochondrial function and SGK1.
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Author Response
Reviewer #1 (Public Review):
Kim et al. demonstrated biphasic roles of ERK-MAPK-mTOR pathway in osteoblast differentiation. They first showed the administration of the MEK inhibitor trametinib increased bone formation and prevented bone loss in OVX mice. They also confirmed the effect of MEK inhibition on late phases of osteoblast differentiation in the culture of human bone marrow-derived mesenchymal stromal cells (hBMSCs). They then focused on the action of ERK-MAPK pathway on the late phase. Indeed, deletion of MEK1 and MEK2 in mature osteoblasts and osteocytes (Dmp1-cre-dKO) led to increased bone mass with augmented osteoblast function, which was also confirmed by an in vitro culture of the mutants' osteoblasts; Ocn-cre-mediated inducible deletion of MEK1 and MEK2 in mature osteoblasts resulted in the similar …
Author Response
Reviewer #1 (Public Review):
Kim et al. demonstrated biphasic roles of ERK-MAPK-mTOR pathway in osteoblast differentiation. They first showed the administration of the MEK inhibitor trametinib increased bone formation and prevented bone loss in OVX mice. They also confirmed the effect of MEK inhibition on late phases of osteoblast differentiation in the culture of human bone marrow-derived mesenchymal stromal cells (hBMSCs). They then focused on the action of ERK-MAPK pathway on the late phase. Indeed, deletion of MEK1 and MEK2 in mature osteoblasts and osteocytes (Dmp1-cre-dKO) led to increased bone mass with augmented osteoblast function, which was also confirmed by an in vitro culture of the mutants' osteoblasts; Ocn-cre-mediated inducible deletion of MEK1 and MEK2 in mature osteoblasts resulted in the similar phenotypes. However, osteocyte apoptosis was increased in Dmp1-cre-dKO. Gene expression profile obtained by RNA-seq supported the mutants' osteoblast phenotypes. Besides osteoblast differentiation-related genes, angiogenic factors were upregulated in the mutants. Conditioned medium of the mutants' osteoblasts enhanced osteogenic potential of mouse BMSCs and in vitro capillary formation of endothelial progenitors. They further found that ERK inhibition augmented glutamine metabolism and mitochondrial function, possibly leading to enhancement of osteoblast function. Lastly, they demonstrated that mTORC2 and its downstream factor SGK1 was involved in the ERK inhibition-mediated osteoblast phenotypes. Based on these data, they propose that the ERK-mTORC2 axis, where ERK inhibits mTORC2, regulates osteoblast differentiation and angiogenesis. Overall this study is well performed, and the manuscript is clearly written.
We thank the reviewer for summarizing and highlighting the significance of our manuscript. We appreciate the reviewer’s constructive suggestions and believe that addressing these points has strengthened the manuscript.
Reviewer #2 (Public Review):
The authors found that the unique role of Erk signaling pathway that inhibited osteoblastogenesis and bone formation at the late stage, while Erk has widely shown to be essential for bone and osteoblast development. These data are also useful for the Readers. In vivo results including OVX experiments and phenotypes of Mek1/2 cKO mice are very interesting and useful information for the bone field, probably for other fields with some interest. The idea to show the mechanism by performing in vitro-based approaches sounds potentially interesting and novel. Conversely, although the authors claim that Erk-mTOR2-SGK1 pathway plays a role in the phenomenon found in these in vivo experiments, the evidence is very weak and preliminary. More appropriate and straightforward approaches and experimental design could strengthen their conclusion. The results shown in the manuscript in this part are still phenomenological. Several important questions were not solved. In particular, the linkage between MEK-Erk and mTOR2-SGK1 with mitochondria is still elusive. The rescue experiments in the cKO mice would be appreciated. Since in vivo and in vitro experiments for the early and late stage of bone formation did not reflect their purpose very much, the authors could re-write the manuscript.
We thank the reviewer for highlighting the significance of our manuscript. We agree that strengthening the mechanistic studies on the ERK-mTORC2-SGK1 pathway is important to strengthen the overall revised manuscript, and have added additional data on this topic to the revised manuscript.
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Evaluation Summary:
This work provides a novel insight into regulation of osteogenesis by ERK-mTOR pathway. The authors proposed that the effect of Erk pathway would be mediated mTOR2-SGK1. The mitochondrial metabolisms appears to be involved in this regulation. This study is well performed, and the manuscript is clearly written.
(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):
Kim et al. demonstrated biphasic roles of ERK-MAPK-mTOR pathway in osteoblast differentiation. They first showed the administration of the MEK inhibitor trametinib increased bone formation and prevented bone loss in OVX mice. They also confirmed the effect of MEK inhibition on late phases of osteoblast differentiation in the culture of human bone marrow-derived mesenchymal stromal cells (hBMSCs). They then focused on the action of ERK-MAPK pathway on the late phase. Indeed, deletion of MEK1 and MEK2 in mature osteoblasts and osteocytes (Dmp1-cre-dKO) led to increased bone mass with augmented osteoblast function, which was also confirmed by an in vitro culture of the mutants' osteoblasts; Ocn-cre-mediated inducible deletion of MEK1 and MEK2 in mature osteoblasts resulted in the similar phenotypes. However, …
Reviewer #1 (Public Review):
Kim et al. demonstrated biphasic roles of ERK-MAPK-mTOR pathway in osteoblast differentiation. They first showed the administration of the MEK inhibitor trametinib increased bone formation and prevented bone loss in OVX mice. They also confirmed the effect of MEK inhibition on late phases of osteoblast differentiation in the culture of human bone marrow-derived mesenchymal stromal cells (hBMSCs). They then focused on the action of ERK-MAPK pathway on the late phase. Indeed, deletion of MEK1 and MEK2 in mature osteoblasts and osteocytes (Dmp1-cre-dKO) led to increased bone mass with augmented osteoblast function, which was also confirmed by an in vitro culture of the mutants' osteoblasts; Ocn-cre-mediated inducible deletion of MEK1 and MEK2 in mature osteoblasts resulted in the similar phenotypes. However, osteocyte apoptosis was increased in Dmp1-cre-dKO. Gene expression profile obtained by RNA-seq supported the mutants' osteoblast phenotypes. Besides osteoblast differentiation-related genes, angiogenic factors were upregulated in the mutants. Conditioned medium of the mutants' osteoblasts enhanced osteogenic potential of mouse BMSCs and in vitro capillary formation of endothelial progenitors. They further found that ERK inhibition augmented glutamine metabolism and mitochondrial function, possibly leading to enhancement of osteoblast function. Lastly, they demonstrated that mTORC2 and its downstream factor SGK1 was involved in the ERK inhibition-mediated osteoblast phenotypes. Based on these data, they propose that the ERK-mTORC2 axis, where ERK inhibits mTORC2, regulates osteoblast differentiation and angiogenesis.
Overall this study is well performed, and the manuscript is clearly written.
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Reviewer #2 (Public Review):
The authors found that the unique role of Erk signaling pathway that inhibited osteoblastogenesis and bone formation at the late stage, while Erk has widely shown to be essential for bone and osteoblast development. These data are also useful for the Readers. In vivo results including OVX experiments and phenotypes of Mek1/2 cKO mice are very interesting and useful information for the bone field, probably for other fields with some interest. The idea to show the mechanism by performing in vitro-based approaches sounds potentially interesting and novel. Conversely, although the authors claim that Erk-mTOR2-SGK1 pathway plays a role in the phenomenon found in these in vivo experiments, the evidence is very weak and preliminary. More appropriate and straightforward approaches and experimental design could …
Reviewer #2 (Public Review):
The authors found that the unique role of Erk signaling pathway that inhibited osteoblastogenesis and bone formation at the late stage, while Erk has widely shown to be essential for bone and osteoblast development. These data are also useful for the Readers. In vivo results including OVX experiments and phenotypes of Mek1/2 cKO mice are very interesting and useful information for the bone field, probably for other fields with some interest. The idea to show the mechanism by performing in vitro-based approaches sounds potentially interesting and novel. Conversely, although the authors claim that Erk-mTOR2-SGK1 pathway plays a role in the phenomenon found in these in vivo experiments, the evidence is very weak and preliminary. More appropriate and straightforward approaches and experimental design could strengthen their conclusion. The results shown in the manuscript in this part are still phenomenological. Several important questions were not solved. In particular, the linkage between MEK-Erk and mTOR2-SGK1 with mitochondria is still elusive. The rescue experiments in the cKO mice would be appreciated. Since in vivo and in vitro experiments for the early and late stage of bone formation did not reflect their purpose very much, the authors could re-write the manuscript.
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