MMP14 cleaves PTH1R in the chondrocyte-derived osteoblast lineage, curbing signaling intensity for proper bone anabolism
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eLife assessment
In this study, the authors presented the novel findings that PTH signaling plays a significant role in bone formation in hypertrophic chondrocyte (HC)-derived osteoblasts and MMP14 cleaves PTH1R and inhibits PTH signaling. These studies significantly contribute to our understanding of molecular mechanisms of postnatal bone formation and adult bone remodeling, especially the HC cells in this process. The study was well-designed and well-conducted. The data in this study are convincing and support the conclusion made by the authors.
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Abstract
Bone homeostasis is regulated by hormones such as parathyroid hormone (PTH). While PTH can stimulate osteo-progenitor expansion and bone synthesis, how the PTH-signaling intensity in progenitors is controlled is unclear. Endochondral bone osteoblasts arise from perichondrium-derived osteoprogenitors and hypertrophic chondrocytes (HC). We found, via single-cell transcriptomics, that HC-descendent cells activate membrane-type 1 metalloproteinase 14 (MMP14) and the PTH pathway as they transition to osteoblasts in neonatal and adult mice. Unlike Mmp14 global knockouts, postnatal day 10 (p10) HC lineage-specific Mmp14 null mutants (Mmp14ΔHC) produce more bone. Mechanistically, MMP14 cleaves the extracellular domain of PTH1R, dampening PTH signaling, and consistent with the implied regulatory role, in Mmp14ΔHC mutants, PTH signaling is enhanced. We found that HC-derived osteoblasts contribute ~50% of osteogenesis promoted by treatment with PTH 1–34, and this response was amplified in Mmp14ΔHC. MMP14 control of PTH signaling likely applies also to both HC- and non-HC-derived osteoblasts because their transcriptomes are highly similar. Our study identifies a novel paradigm of MMP14 activity-mediated modulation of PTH signaling in the osteoblast lineage, contributing new insights into bone metabolism with therapeutic significance for bone-wasting diseases.
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Author Response
Reviewer #2 (Public Review):
This is a highly interesting paper that provides important insights into the understanding of how HC-derived osteoblasts contribute to trabecular bone formation. Using single-cell transcriptomics, the authors found that HC descendent cells activate MMP14 and the PTH pathway as they transition to osteoblasts in neonatal and adult mice. They further demonstrate that HC lineage-specific Mmp14 null mutants (Mmp14ΔHC) produce more bone. By performing a panel of elegant in vitro studies, the authors show that MMP14 cleaves the extracellular domain of PTH1R, dampening PTH signaling. The authors provide more in vivo evidence showing that HC-derived osteogenic cells respond to PTH which is enhanced in Mmp14ΔHC. Generally, this is a very well-performed study that may contribute important novel …
Author Response
Reviewer #2 (Public Review):
This is a highly interesting paper that provides important insights into the understanding of how HC-derived osteoblasts contribute to trabecular bone formation. Using single-cell transcriptomics, the authors found that HC descendent cells activate MMP14 and the PTH pathway as they transition to osteoblasts in neonatal and adult mice. They further demonstrate that HC lineage-specific Mmp14 null mutants (Mmp14ΔHC) produce more bone. By performing a panel of elegant in vitro studies, the authors show that MMP14 cleaves the extracellular domain of PTH1R, dampening PTH signaling. The authors provide more in vivo evidence showing that HC-derived osteogenic cells respond to PTH which is enhanced in Mmp14ΔHC. Generally, this is a very well-performed study that may contribute important novel aspects to the field.
I have the following issues for the authors to address:
- The novel mechanism identified in this study (i.e. MMP14-induced PTH1R cleavage) is intriguing. It is unclear how specific this pathway is in the transition of HCs to osteoblasts. Are other MMPs besides MMP14 involved in the PTH1R cleavage? Is PTH1R the only substrate of MMP14?
Thank you for your interest in our findings. ADAMs are known to cleave various transmembrane proteins such as RANKL. As described in supplementary fFgure 4A we tested A Disintegrin And Metalloproteinase (ADAMs) for their potential ability to cleave PTH1R. We did not find that ADAM10, 15, 17 could cleave PTH1R. The lack of the cleaved PTH1R peptide in extracts isolated from osteoblasts isolated from MMP 14 null bones (New Fig. 3E) suggest that there is not another major MMP that cleaves PTH1R. In regard to other substrates that are cleaved by MMP14 – we do review these in the manuscript and the possibility that the phenotype is contributed by deficiency in other substrates.
- Would it be possible for the authors to detect the truncated PTH1R fragment(s) from the conditioned medium prepared from either 293T or osteoblast culture?
We tried to detect whether there could be PTH1R cleaved fragment in cultured medium by western blot of PCA precipitates of cultured medium. We could not detect any free peptide using anti-Flag or anti-HA antibody. It has been reported the ligand binding domain are linked by disulphide bond in vivo, therefore cleavage of PTH1R at the unstructured loop domain does not necessarily imply a release of cleaved fragment.
- The finding that HC-descendants persist and contribute to the anabolic response to PTH in aged mice is interesting. Have the authors examined the changes in MMP14 expression in bone with age and in response to PTH treatment?
Thank you for your question, we added additional data showing induction of MMP14 expression upon PTH treatment in Figure 7—figure supplement 1. It has also been published that PTH stimulation increased MMP14 expression in osteocytes (1).
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eLife assessment
In this study, the authors presented the novel findings that PTH signaling plays a significant role in bone formation in hypertrophic chondrocyte (HC)-derived osteoblasts and MMP14 cleaves PTH1R and inhibits PTH signaling. These studies significantly contribute to our understanding of molecular mechanisms of postnatal bone formation and adult bone remodeling, especially the HC cells in this process. The study was well-designed and well-conducted. The data in this study are convincing and support the conclusion made by the authors.
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Reviewer #1 (Public Review):
Recent studies indicate that osteoblasts formed during endochondral bone formation have arisen from perichondrium-derived osteoprogenitors and hypertrophic chondrocytes (HC). In this study, through a single-cell transcriptomics approach, the authors found that HC descendent cells activate MMP14 and the PTH pathway as they transition to osteoblasts at postnatal and adult stages. HC-specific Mmp14 knockout mice had increased bone mass phenotype. The authors found that MMP14 cleaves the extracellular domain of PTH1R and inhibits PTH signaling. They also found that HC-derived osteoblasts contribute about 50% of osteogenesis promoted by the treatment with PTH 1-34 and this response was amplified in Mmp14 knockout mice. MMP14 controls PTH signaling through both HC- and non-HC-derived osteoblasts. The authors …
Reviewer #1 (Public Review):
Recent studies indicate that osteoblasts formed during endochondral bone formation have arisen from perichondrium-derived osteoprogenitors and hypertrophic chondrocytes (HC). In this study, through a single-cell transcriptomics approach, the authors found that HC descendent cells activate MMP14 and the PTH pathway as they transition to osteoblasts at postnatal and adult stages. HC-specific Mmp14 knockout mice had increased bone mass phenotype. The authors found that MMP14 cleaves the extracellular domain of PTH1R and inhibits PTH signaling. They also found that HC-derived osteoblasts contribute about 50% of osteogenesis promoted by the treatment with PTH 1-34 and this response was amplified in Mmp14 knockout mice. MMP14 controls PTH signaling through both HC- and non-HC-derived osteoblasts. The authors concluded that they have identified a novel mechanism of MMP14-mediated PTH signaling in the osteoblast lineage cells.
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Reviewer #2 (Public Review):
This is a highly interesting paper that provides important insights into the understanding of how HC-derived osteoblasts contribute to trabecular bone formation. Using single-cell transcriptomics, the authors found that HC descendent cells activate MMP14 and the PTH pathway as they transition to osteoblasts in neonatal and adult mice. They further demonstrate that HC lineage-specific Mmp14 null mutants (Mmp14ΔHC) produce more bone. By performing a panel of elegant in vitro studies, the authors show that MMP14 cleaves the extracellular domain of PTH1R, dampening PTH signaling. The authors provide more in vivo evidence showing that HC-derived osteogenic cells respond to PTH which is enhanced in Mmp14ΔHC. Generally, this is a very well-performed study that may contribute important novel aspects to the field.
I …
Reviewer #2 (Public Review):
This is a highly interesting paper that provides important insights into the understanding of how HC-derived osteoblasts contribute to trabecular bone formation. Using single-cell transcriptomics, the authors found that HC descendent cells activate MMP14 and the PTH pathway as they transition to osteoblasts in neonatal and adult mice. They further demonstrate that HC lineage-specific Mmp14 null mutants (Mmp14ΔHC) produce more bone. By performing a panel of elegant in vitro studies, the authors show that MMP14 cleaves the extracellular domain of PTH1R, dampening PTH signaling. The authors provide more in vivo evidence showing that HC-derived osteogenic cells respond to PTH which is enhanced in Mmp14ΔHC. Generally, this is a very well-performed study that may contribute important novel aspects to the field.
I have the following issues for the authors to address:
1. The novel mechanism identified in this study (i.e. MMP14-induced PTH1R cleavage) is intriguing. It is unclear how specific this pathway is in the transition of HCs to osteoblasts. Are other MMPs besides MMP14 involved in the PTH1R cleavage? Is PTH1R the only substrate of MMP14?
2. Would it be possible for the authors to detect the truncated PTH1R fragment(s) from the conditioned medium prepared from either 293T or osteoblast culture?
3. The finding that HC-descendants persist and contribute to the anabolic response to PTH in aged mice is interesting. Have the authors examined the changes in MMP14 expression in bone with age and in response to PTH treatment? -
Reviewer #3 (Public Review):
In this manuscript, Chu and colleagues first studied the differentiation of hypertrophic chondrocytes into osteoblasts using lineage tracing and single-cell transcriptomics on dissociated bone tissues. In analyzing these data, they identified MMP14 as upregulated in immature osteoblasts derived from hypertrophic chondrocytes. This observation prompted them to study the relationship between MMP14 and signals that regulate osteoblast differentiation such as a parathyroid hormone. Interestingly, MMP14 was found to cleave the ectodomain of the PTH receptor and blunt its signaling activity. Accordingly, MMP14 deficiency in these cells augmented PTH-induced bone anabolism.
This work builds upon multiple previous studies demonstrating that a subset of hypertrophic chondrocytes (or, at least, cells marked by …
Reviewer #3 (Public Review):
In this manuscript, Chu and colleagues first studied the differentiation of hypertrophic chondrocytes into osteoblasts using lineage tracing and single-cell transcriptomics on dissociated bone tissues. In analyzing these data, they identified MMP14 as upregulated in immature osteoblasts derived from hypertrophic chondrocytes. This observation prompted them to study the relationship between MMP14 and signals that regulate osteoblast differentiation such as a parathyroid hormone. Interestingly, MMP14 was found to cleave the ectodomain of the PTH receptor and blunt its signaling activity. Accordingly, MMP14 deficiency in these cells augmented PTH-induced bone anabolism.
This work builds upon multiple previous studies demonstrating that a subset of hypertrophic chondrocytes (or, at least, cells marked by collagen X Cre strategies) can become osteoblasts. The use of lineage tracing to try to divide osteoblasts into those derived from HCs or other progenitors is interesting, although technical challenges are present in data interpretation. The study then pivots dramatically into loosely-connected mechanistic studies investigating links between MMP14 (identified from their single-cell RNA-seq studies) and the PTH receptor. Gaps exist in the logic linking this work to the beginning of the paper, and major questions remain about MMP14-mediated PTH receptor cleavage. The work then returns to in vivo studies investigating the skeletal and cellular phenotype of PTH-treated mice where MMP14 is deleted using collagen X Cre.
While several interesting threads are suggested by these findings, the scope of the work is quite broad and it is difficult to appreciate the direct relationship between some of the findings that are presented in successive figures. GPCR cleavage by an MMP is exciting and interesting. However, the cleavage patterns observed in vitro do not match the PTH receptor fragments noted in vivo. Moreover, much remains to be described regarding differences in PTH efficacy in cells with and without MMP14. Of course, the possibility remains that MMP14 targets other than the PTH receptor contribute to the phenotypes that are observed in mice.
This work adds to an already-large body of evidence demonstrating that collagen X-labeled cells contribute to the osteoblast pool. The use of single-cell RNA-seq here is appealing and demonstrates the heterogeneity of collagen X-labeled cells and their descendants for the first time. The scRNAseq data will be useful for the entire bone biology community. In addition, a comparison between global and ColX-mediated MMP14 deletion is well done and of interest. Overall, my impression of the impact of the work is mixed. The most novel/exciting finding here is that MMP14 cleaves the PTH receptor and regulates its activity: the evidence supporting this new finding is incomplete, and the other data presented on hypertrophic chondrocyte differentiation may be viewed as a distraction to the central message of this manuscript.
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