Transferrin receptor 1-mediated iron uptake regulates bone mass in mice via osteoclast mitochondria and cytoskeleton
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Evaluation Summary:
The authors present a very well written manuscript addressing an important unknown in bone homeostasis, aiming to understand the mechanism of iron mediated effects on bone, findings of novel significance that are of interest to basic iron biologists, bone biologists, experts in mitochondrial respiration, and endocrinologists. This is the first study to show that Tfr1 is important for iron uptake in vivo and for proper osteoclast function. Mechanistically, Tfr1-mediated iron uptake is important for mitochondrial function and cytoskeleton organization, which is important for bone resorption. Overall, this study adds important information regarding the role of Tfr1 and iron metabolism in osteoclasts.
(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 #2 agreed to share their name with the authors.)
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Abstract
Increased intracellular iron spurs mitochondrial biogenesis and respiration to satisfy high-energy demand during osteoclast differentiation and bone-resorbing activities. Transferrin receptor 1 (Tfr1) mediates cellular iron uptake through endocytosis of iron-loaded transferrin, and its expression increases during osteoclast differentiation. Nonetheless, the precise functions of Tfr1 and Tfr1-mediated iron uptake in osteoclast biology and skeletal homeostasis remain incompletely understood. To investigate the role of Tfr1 in osteoclast lineage cells in vivo and in vitro, we crossed Tfrc (encoding Tfr1)-floxed mice with Lyz2 (LysM)- Cre and Cathepsin K ( Ctsk )-Cre mice to generate Tfrc conditional knockout mice in myeloid osteoclast precursors (Tfr1 ΔLysM ) or differentiated osteoclasts (Tfr1 ΔCtsk ), respectively. Skeletal phenotyping by µCT and histology unveiled a significant increase in trabecular bone mass with normal osteoclast number in long bones of 10-week-old young and 6-month-old adult female but not male Tfr1 ΔLysM mice. Although high trabecular bone volume in long bones was observed in both male and female Tfr1 ΔCtsk mice, this phenotype was more pronounced in female knockout mice. Consistent with this gender-dependent phenomena, estrogen deficiency induced by ovariectomy decreased trabecular bone mass in Tfr1 ΔLysM mice. Mechanistically, disruption of Tfr1 expression attenuated mitochondrial metabolism and cytoskeletal organization in mature osteoclasts in vitro by attenuating mitochondrial respiration and activation of the Src-Rac1-WAVE regulatory complex axis, respectively, leading to decreased bone resorption with little impact on osteoclast differentiation. These results indicate that Tfr1-mediated iron uptake is specifically required for osteoclast function and is indispensable for bone remodeling in a gender-dependent manner.
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Evaluation Summary:
The authors present a very well written manuscript addressing an important unknown in bone homeostasis, aiming to understand the mechanism of iron mediated effects on bone, findings of novel significance that are of interest to basic iron biologists, bone biologists, experts in mitochondrial respiration, and endocrinologists. This is the first study to show that Tfr1 is important for iron uptake in vivo and for proper osteoclast function. Mechanistically, Tfr1-mediated iron uptake is important for mitochondrial function and cytoskeleton organization, which is important for bone resorption. Overall, this study adds important information regarding the role of Tfr1 and iron metabolism in osteoclasts.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also …
Evaluation Summary:
The authors present a very well written manuscript addressing an important unknown in bone homeostasis, aiming to understand the mechanism of iron mediated effects on bone, findings of novel significance that are of interest to basic iron biologists, bone biologists, experts in mitochondrial respiration, and endocrinologists. This is the first study to show that Tfr1 is important for iron uptake in vivo and for proper osteoclast function. Mechanistically, Tfr1-mediated iron uptake is important for mitochondrial function and cytoskeleton organization, which is important for bone resorption. Overall, this study adds important information regarding the role of Tfr1 and iron metabolism in osteoclasts.
(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 #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Zhao et al present a very well written manuscript focused on exploring the effect and mechanism of TFR1 loss in osteoclast lineage cells. This work is unique with no precedent in or expectation of whether and how iron deficiency via TFR1 loss affects bone homeostasis. In the current manuscript, the authors focus specifically on osteoclast-lineage cells and demonstrate decreased osteoclast function via impact on mitochondrial ROS-dependent function and respiration leading to a resorption defect and in vivo increase in bone volume in TFR1 fl/fl-Ctsk-Cre mice. The work is compelling in many ways and yet several comments are warranted for clarification. Specifically, it is unclear what the added value of the LysM-cre model is in the current work and appears to only distract from the main focus of the manuscript, …
Reviewer #1 (Public Review):
Zhao et al present a very well written manuscript focused on exploring the effect and mechanism of TFR1 loss in osteoclast lineage cells. This work is unique with no precedent in or expectation of whether and how iron deficiency via TFR1 loss affects bone homeostasis. In the current manuscript, the authors focus specifically on osteoclast-lineage cells and demonstrate decreased osteoclast function via impact on mitochondrial ROS-dependent function and respiration leading to a resorption defect and in vivo increase in bone volume in TFR1 fl/fl-Ctsk-Cre mice. The work is compelling in many ways and yet several comments are warranted for clarification. Specifically, it is unclear what the added value of the LysM-cre model is in the current work and appears to only distract from the main focus of the manuscript, that effects at the mature osteoclast stage are most robust. Furthermore, the authors do not reconcile whether effects on the mitochondrial (Fig 9 and 10) are reversed with the addition of hemin. In addition, it is not clear why TFR1-fl-LysM-cre mice are used rather than TFR1-fl-Ctsk-cre in the in vitro assay performed; it remains a possibility that the results would be altered if the TFR1 loss occurred later in differentiation. Finally, the data on Hem1 overexpression is interesting but all appropriate controls are not included to definitively support a conclusion that TFR1-meadiated effects on osteoclasts are a consequence of destabilizing WRC complex.
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Reviewer #2 (Public Review):
The authors investigated the role of the transferrin receptor 1 (Tfr1)-mediated iron uptake in osteoclasts and its impact on bone homeostasis and found that conditional deletion of Tfr1 in osteoclast lineage cells resulted in increased bone mass primarily in the long bones of female mice. In addition, the authors show that genetic disruption of Tfr1 alters mitochondrial metabolism and the osteoclast cytoskeletal organization. The strength of the manuscript is the novelty i.e. the first to assess the physiological contribution of Tfr1 in osteoclasts using two complementary genetically-modified mouse models that conditionally target osteoclast-progenitor cells and mature osteoclasts. The weakness of the study is that the skeletal phenotyping is restricted to a single time point i.e. 10-weeks of age and …
Reviewer #2 (Public Review):
The authors investigated the role of the transferrin receptor 1 (Tfr1)-mediated iron uptake in osteoclasts and its impact on bone homeostasis and found that conditional deletion of Tfr1 in osteoclast lineage cells resulted in increased bone mass primarily in the long bones of female mice. In addition, the authors show that genetic disruption of Tfr1 alters mitochondrial metabolism and the osteoclast cytoskeletal organization. The strength of the manuscript is the novelty i.e. the first to assess the physiological contribution of Tfr1 in osteoclasts using two complementary genetically-modified mouse models that conditionally target osteoclast-progenitor cells and mature osteoclasts. The weakness of the study is that the skeletal phenotyping is restricted to a single time point i.e. 10-weeks of age and mechanistic evidence linking Tfr1-mediated iron uptake to regulation of the osteoclast cytoskeleton remains preliminary. Overall, this study offers the first detailed phenotypic assessment of the skeleton of Tfr1-deficient mice and provides new insights into the importance of iron uptake for osteoclast function during bone turnover.
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Reviewer #3 (Public Review):
Das et al. aimed to investigate the role of Tfr1 for iron uptake in osteoclasts. They have used two in vivo models to knockout Tfr1 in osteoclast precursors (myeloid cells) and mature osteoclasts and show that Tfr1 is an important iron uptake receptor in mature osteoclasts. In particular, iron is important for mitochondrial function and cytoskeletal reorganization.The strength of this investigation is the rigorous use of controls. Also, their use of different ages and Tfr1-knockout strains adds confidence to their conclusions. A weakness is the rather superficial characterization of osteoclastogenesis, which could be done more accurately to really pin-down at which stage of differentiation Tfr1 is particularly required.
Overall, this study adds important information regarding the role of Tfr1 and iron …
Reviewer #3 (Public Review):
Das et al. aimed to investigate the role of Tfr1 for iron uptake in osteoclasts. They have used two in vivo models to knockout Tfr1 in osteoclast precursors (myeloid cells) and mature osteoclasts and show that Tfr1 is an important iron uptake receptor in mature osteoclasts. In particular, iron is important for mitochondrial function and cytoskeletal reorganization.The strength of this investigation is the rigorous use of controls. Also, their use of different ages and Tfr1-knockout strains adds confidence to their conclusions. A weakness is the rather superficial characterization of osteoclastogenesis, which could be done more accurately to really pin-down at which stage of differentiation Tfr1 is particularly required.
Overall, this study adds important information regarding the role of Tfr1 and iron metabolism in osteoclasts and suggests that targeting iron-related pathways may be a way forward to therapeutically treat bone loss.
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