C-type natriuretic peptide facilitates autonomic Ca2+ entry in growth plate chondrocytes for stimulating bone growth
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
The manuscript provides novel data to support the role of CNP (C Type Natriuretic peptide) in the proliferation and stimulation of growth plate chondrocytes for development of bone. The methods used are innovative and the data provided support the overall hypothesis of the study presented in the paper, which should be of broad interest to bone and cartilage biologists.
(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. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
The growth plates are cartilage tissues found at both ends of developing bones, and vital proliferation and differentiation of growth plate chondrocytes are primarily responsible for bone growth. C-type natriuretic peptide (CNP) stimulates bone growth by activating natriuretic peptide receptor 2 (NPR2) which is equipped with guanylate cyclase on the cytoplasmic side, but its signaling pathway is unclear in growth plate chondrocytes. We previously reported that transient receptor potential melastatin-like 7 (TRPM7) channels mediate intermissive Ca 2+ influx in growth plate chondrocytes, leading to activation of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) for promoting bone growth. In this report, we provide evidence from experiments using mutant mice, indicating a functional link between CNP and TRPM7 channels. Our pharmacological data suggest that CNP-evoked NPR2 activation elevates cellular cGMP content and stimulates big-conductance Ca 2+ -dependent K + (BK) channels as a substrate for cGMP-dependent protein kinase (PKG). BK channel-induced hyperpolarization likely enhances the driving force of TRPM7-mediated Ca 2+ entry and seems to accordingly activate CaMKII. Indeed, ex vivo organ culture analysis indicates that CNP-facilitated bone growth is abolished by chondrocyte-specific Trpm7 gene ablation. The defined CNP signaling pathway, the NPR2-PKG-BK channel–TRPM7 channel–CaMKII axis, likely pinpoints promising target proteins for developing new therapeutic treatments for divergent growth disorders.
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Author Response
Reviewer #1 (Public Review):
Weaknesses:
- The paper lacks some clarification in some terms used in the paper such as "round chondrocytes". Additional histological data on the organ cultures would provide supporting evidence on the role of CNP in chondrocytes.
In bone research fields, growth plate chondrocytes are generally classified into three major cell types; round, columnar, and hypertrophic chondrocytes, and “round chondrocytes” is generally used for specifying growth plate chondrocytes. We have briefly explained cells assessed in the Materials and Methods section of the revised manuscript.
In response to the comment, we histologically analyzed cultured CNP-treated bones from the chondrocyte specific Trpm7-knockout (Trpm7fl/fl, 11Enh-Cre+/−) and control (Trpm7fl/fl, 11Enh-Cre−/−) mice. The new data obtained …
Author Response
Reviewer #1 (Public Review):
Weaknesses:
- The paper lacks some clarification in some terms used in the paper such as "round chondrocytes". Additional histological data on the organ cultures would provide supporting evidence on the role of CNP in chondrocytes.
In bone research fields, growth plate chondrocytes are generally classified into three major cell types; round, columnar, and hypertrophic chondrocytes, and “round chondrocytes” is generally used for specifying growth plate chondrocytes. We have briefly explained cells assessed in the Materials and Methods section of the revised manuscript.
In response to the comment, we histologically analyzed cultured CNP-treated bones from the chondrocyte specific Trpm7-knockout (Trpm7fl/fl, 11Enh-Cre+/−) and control (Trpm7fl/fl, 11Enh-Cre−/−) mice. The new data obtained clearly indicate that CNP treatments extend the columnar chondrocytic zones in control bones but not in chondrocyte-specific Trpm7-knockout bones (Figure 7 in the revised manuscript). We further analyzed in detail CNP-treated metatarsal bones from wild-type mice. CNP treatments consistently expanded the columnar chondrocytic zones but did not affect the cell densities (Figure 7-figure supplement 1 in the revised manuscript). Moreover, CNP-promoted extracellular matrix production seemed to contribute largely to the extension of columnar chondrocyte zone toward bone outgrowth stimulation.
- The authors also generated NPR2 chondrocytes-specific null mouse mode. It would be interesting to include some data on the phenotype of these mice by micro-CT, histology, etc. More specific details are provided below.
Overview of histological analysis in chondrocyte-specific Npr2-knockout (Npr2fl/fl, Col2a1-Cre+/−) mice has been previously reported (Nakao K., et al., Sci Rep., 5;10554, 2015). Since femoral bones from E17.5 mice were too fragile to analyze using micro-CT, we performed von Kossa-staining of femoral bones from the chondrocyte-specific Npr2-knockout (Npr2fl/fl, Col2a1-Cre+/−) and control (Npr2fl/fl, Col2a1-Cre−/−) mice instead. As reasonably expected, femoral bones from the knockout mice exhibited insufficient mineralization.
The reviewer believe the authors achieved their aims, while some additional supporting data and clarifications are needed.
The overall summary of the data provided is supportive, methods utilized are well justified, again, additional histological data would support the overall summary.
Data generated and presented in the report will enhance our understanding on the role of CNP and possible utilization of novel therapeutic targets for CNP for the treatment of bone growth diseases.
We thank the reviewer for his valuable and helpful comments.
Reviewer #2 (Public Review):
Strength:
- Experiments are well designed, performed with proper control and conclusion drawn from data is highly convincing.
- Valid animal model (floxed mice) using chondrocyte specific deletion of gene were used to show in vivo effect of gene deletion on bone formation.
- Preclinical model of OA were used in vivo using mice as an experimental animal, OA progression were characterized using established grading system.
- All the Western blots are shown along with densitometric quantification.
- Ex vivo molecular mechanism is also provided which added strength in the manuscript.
Methods, results and data interpretation:
- Methods section is adequate and describes enough details to replicate in independent study.
- Relevant statistics are used to infer conclusion form data.
- There is no objective error in presenting the data, conclusions drawn from experiments are convincing.
We thank the reviewer for the encouraging comments.
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Evaluation Summary:
The manuscript provides novel data to support the role of CNP (C Type Natriuretic peptide) in the proliferation and stimulation of growth plate chondrocytes for development of bone. The methods used are innovative and the data provided support the overall hypothesis of the study presented in the paper, which should be of broad interest to bone and cartilage biologists.
(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. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
The papers addresses an important aspects of CNP function in chondrocytes and its signaling pathway.
Strengths:
The hypothesis proposed for this paper. The quality of the data presented and the writing style of the paper.
Weaknesses:
The paper lacks some clarification in some terms used in the paper such as "round chondrocytes". Additional histological data on the organ cultures would provide supporting evidence on the role of CNP in chondrocytes.
The authors also generated NPR2 chondrocyte-specific null mice. It would be interesting to include some data on the phenotype of these mice by micro-CT, histology, etc.
The reviewer believe the authors achieved their aims, while some additional supporting data and clarifications are needed.
The overall summary of the data provided is supportive, methods utilized …
Reviewer #1 (Public Review):
The papers addresses an important aspects of CNP function in chondrocytes and its signaling pathway.
Strengths:
The hypothesis proposed for this paper. The quality of the data presented and the writing style of the paper.
Weaknesses:
The paper lacks some clarification in some terms used in the paper such as "round chondrocytes". Additional histological data on the organ cultures would provide supporting evidence on the role of CNP in chondrocytes.
The authors also generated NPR2 chondrocyte-specific null mice. It would be interesting to include some data on the phenotype of these mice by micro-CT, histology, etc.
The reviewer believe the authors achieved their aims, while some additional supporting data and clarifications are needed.
The overall summary of the data provided is supportive, methods utilized are well justified, again, additional histological data would support the overall summary.
Data generated and presented in the report will enhance our understanding on the role of CNP and possible utilization of novel therapeutic targets for CNP for the treatment of bone growth diseases.
-
Reviewer #2 (Public Review):
Miyazaki et al demonstrated the role of CNP (C Type Natriuretic peptide) in the proliferation and stimulation of growth plate chondrocytes for development of bone. The author dissected the signaling axis involved in CNP mediated bone growth and discover the role of Ca+2 mediated signaling by identifying the NPR2-PKG-BK-TRPM7-CaMKII axis in bone growth. This study provides an exciting data in the field of bone biology and provided a new layer of regulation for bone growth. Overall study design is logical, and experiments were well performed and organized. Adequate number of animals were used in study and conclusions drawn from experiments are convincing.
Strength:
- Experiments are well designed, performed with proper control and conclusion drawn from data is highly convincing.
- Valid animal model (floxed mice) …
Reviewer #2 (Public Review):
Miyazaki et al demonstrated the role of CNP (C Type Natriuretic peptide) in the proliferation and stimulation of growth plate chondrocytes for development of bone. The author dissected the signaling axis involved in CNP mediated bone growth and discover the role of Ca+2 mediated signaling by identifying the NPR2-PKG-BK-TRPM7-CaMKII axis in bone growth. This study provides an exciting data in the field of bone biology and provided a new layer of regulation for bone growth. Overall study design is logical, and experiments were well performed and organized. Adequate number of animals were used in study and conclusions drawn from experiments are convincing.
Strength:
- Experiments are well designed, performed with proper control and conclusion drawn from data is highly convincing.
- Valid animal model (floxed mice) using chondrocyte specific deletion of gene were used to show in vivo effect of gene deletion on bone formation.
- Preclinical model of OA were used in vivo using mice as an experimental animal, OA progression were characterized using established grading system.
- All the Western blots are shown along with densitometric quantification.
- Ex vivo molecular mechanism is also provided which added strength in the manuscript.
Methods, results and data interpretation:
- Methods section is adequate and describes enough details to replicate in independent study.
- Relevant statistics are used to infer conclusion form data.
- There is no objective error in presenting the data, conclusions drawn from experiments are convincing.
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