PTH regulates osteogenesis and suppresses adipogenesis through Zfp467 in a feed-forward, PTH1R-cyclic AMP-dependent manner
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eLife assessment
The study provides evidence that the hormone PTH increases bone mass by, at least in part, regulating the factor Zfp467. In turn, Zfp67 controls expression of the receptor for PTH, thus creating a feedback loop that overall augments bone mass. The findings are novel and of potential great interest. Overall, the study is of interest to a broad audience and significant as it unveils a novel feedback loop involving PTH, a critical endocrine regulator of calcium, phosphate, and bone mass.
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Abstract
Conditional deletion of the PTH1R in mesenchymal progenitors reduces osteoblast differentiation, enhances marrow adipogenesis, and increases zinc finger protein 467 ( Zfp467 ) expression. In contrast, genetic loss of Zfp467 increased Pth1r expression and shifts mesenchymal progenitor cell fate toward osteogenesis and higher bone mass. PTH1R and ZFP467 could constitute a feedback loop that facilitates PTH-induced osteogenesis and that conditional deletion of Zfp467 in osteogenic precursors would lead to high bone mass in mice. Prrx1Cre; Zfp467 fl/fl but not AdipoqCre; Zfp467 fl/fl mice exhibit high bone mass and greater osteogenic differentiation similar to the Zfp467 -/- mice. qPCR results revealed that PTH suppressed Zfp467 expression primarily via the cyclic AMP/PKA pathway. Not surprisingly, PKA activation inhibited the expression of Zfp467 and gene silencing of Pth1r caused an increase in Zfp467 mRNA transcription. Dual fluorescence reporter assays and confocal immunofluorescence demonstrated that genetic deletion of Zfp467 resulted in higher nuclear translocation of NFκB1 that binds to the P2 promoter of the Pth1r and increased its transcription. As expected, Zfp467 -/- cells had enhanced production of cyclic AMP and increased glycolysis in response to exogenous PTH. Additionally, the osteogenic response to PTH was also enhanced in Zfp467 -/- COBs, and the pro-osteogenic effect of Zfp467 deletion was blocked by gene silencing of Pth1r or a PKA inhibitor. In conclusion, our findings suggest that loss or PTH1R-mediated repression of Zfp467 results in a pathway that increases Pth1r transcription via NFκB1 and thus cellular responsiveness to PTH/PTHrP, ultimately leading to enhanced bone formation.
Article activity feed
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eLife assessment
The study provides evidence that the hormone PTH increases bone mass by, at least in part, regulating the factor Zfp467. In turn, Zfp67 controls expression of the receptor for PTH, thus creating a feedback loop that overall augments bone mass. The findings are novel and of potential great interest. Overall, the study is of interest to a broad audience and significant as it unveils a novel feedback loop involving PTH, a critical endocrine regulator of calcium, phosphate, and bone mass.
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Reviewer #1 (Public Review):
Overall, this manuscript by Liu et al. provides a largely convincing mechanism for both how Zfp467 regulates osteoblast differentiation and how PTH1R expression and function in osteoblast-lineage cells is regulated at the transcriptional level, finding that NF-kB (RelB/p50) regulates PTH1R expression downstream of Zfp467. PTH1R expression and activity in turn is enhanced in Zfp467-deficient osteoblasts. In turn, PTH signaling regulates Zfp467 expression through PKA activity. In particular, the new findings on mechanisms of regulating PTH1R expression and evidence that this in turn impacts osteoblast differentiation are felt to be of broad interest and importance. The approach used is felt to be largely sound. Areas of major concern are few and relate mostly to better fleshing out how the NF-kB pathway is …
Reviewer #1 (Public Review):
Overall, this manuscript by Liu et al. provides a largely convincing mechanism for both how Zfp467 regulates osteoblast differentiation and how PTH1R expression and function in osteoblast-lineage cells is regulated at the transcriptional level, finding that NF-kB (RelB/p50) regulates PTH1R expression downstream of Zfp467. PTH1R expression and activity in turn is enhanced in Zfp467-deficient osteoblasts. In turn, PTH signaling regulates Zfp467 expression through PKA activity. In particular, the new findings on mechanisms of regulating PTH1R expression and evidence that this in turn impacts osteoblast differentiation are felt to be of broad interest and importance. The approach used is felt to be largely sound. Areas of major concern are few and relate mostly to better fleshing out how the NF-kB pathway is impacted as a part of the molecular pathway implicated here and clarifying some confusion regarding uCT data that appears to be discussed but which this reviewer cannot locate in the figure.
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Reviewer #2 (Public Review):
The authors hypothesized that PTH1R and ZFP467 could constitute a feedback loop that facilitates PTH-induced osteogenesis and that conditional deletion of Zfp467 in osteogenic precursors would lead to high bone mass. Using a number of methods, they have established a regulatory feedback mechanism of this transcription factor and the PTH receptor in osteoblastic precursors as well as showing that PrrxCre deletion of Zfp467 causes an increase in trabecular bone mass, while AdipoCre does not. Nevertheless, they have not established the actual mechanism of action of the transcription factor nor which gene it acts on in the osteoblast. They have mostly achieved their aims and the results partially support their conclusions. However, the work is descriptive and does not address the central issue of how ZFP467 …
Reviewer #2 (Public Review):
The authors hypothesized that PTH1R and ZFP467 could constitute a feedback loop that facilitates PTH-induced osteogenesis and that conditional deletion of Zfp467 in osteogenic precursors would lead to high bone mass. Using a number of methods, they have established a regulatory feedback mechanism of this transcription factor and the PTH receptor in osteoblastic precursors as well as showing that PrrxCre deletion of Zfp467 causes an increase in trabecular bone mass, while AdipoCre does not. Nevertheless, they have not established the actual mechanism of action of the transcription factor nor which gene it acts on in the osteoblast. They have mostly achieved their aims and the results partially support their conclusions. However, the work is descriptive and does not address the central issue of how ZFP467 acts. At present, its impact on the field is limited.
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Reviewer #3 (Public Review):
In this paper the authors report a new pathway by which PTH, by activating PTH1R, increases bone mass. Specifically, they describe that PTH1R and the transcription factor Zfp467 are part of a feedback loop that promotes PTH-induced osteogenesis. By using in vivo mouse models, they indicate that deletion of Zfp467 in osteoblast progenitors increases bone mass and osteogenic differentiation whereas deletion of Zfp467 in adipocytes does not cause any bone phenotype, suggesting that deletion of Zfp467 in mesenchymal progenitors is important for bone mass. With a series of in vivo studies, they demonstrate that PTH suppresses Zfp467 expression via the cyclic AMP-PKA pathway and genetic deletion of Zfp467 causes increased PTH1R transcription by increasing nuclear translocation of p50 and activation of the P2 …
Reviewer #3 (Public Review):
In this paper the authors report a new pathway by which PTH, by activating PTH1R, increases bone mass. Specifically, they describe that PTH1R and the transcription factor Zfp467 are part of a feedback loop that promotes PTH-induced osteogenesis. By using in vivo mouse models, they indicate that deletion of Zfp467 in osteoblast progenitors increases bone mass and osteogenic differentiation whereas deletion of Zfp467 in adipocytes does not cause any bone phenotype, suggesting that deletion of Zfp467 in mesenchymal progenitors is important for bone mass. With a series of in vivo studies, they demonstrate that PTH suppresses Zfp467 expression via the cyclic AMP-PKA pathway and genetic deletion of Zfp467 causes increased PTH1R transcription by increasing nuclear translocation of p50 and activation of the P2 promoter, thus increasing the expression of PTH1R and the cellular responsiveness to PTH.
The strength of the paper are the use of genetically modified animal models, the analysis of both female and male mice and the logic flow of the in vitro data. The weaknesses of the paper are the wrong conclusion that the phenotype of the PrrxCre Zfp467 mice perfectly recapitulates one of the global Zfp467 KO mice, the lack of histomorphometric data showing increased osteoblastogenesis and the missing evidence that the forward feedback loop is relevant to the response to PTH in vivo. The analysis is therefore incomplete. Once those points are addressed, this paper would be of great interest in the bone field.
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