Formation of multinucleated osteoclasts depends on an oxidized species of cell surface associated La protein
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eLife assessment
This manuscript provides an important advance in our understanding of the molecular events that promote osteoclast fusion. Compelling data support the conclusion that an oxidized form of the ubiquitous protein La promotes osteoclast fusion following enrichment at the cell surface of osteoclast progenitors. These data improve our understanding of the processes that regulate bone resorption and will be of broad interest to researchers in the fields of cell biology and musculoskeletal physiology.
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Abstract
The bone-resorbing activity of osteoclasts plays a critical role in the life-long remodeling of our bones that is perturbed in many bone loss diseases. Multinucleated osteoclasts are formed by the fusion of precursor cells, and larger cells - generated by an increased number of cell fusion events - have higher resorptive activity. We find that osteoclast fusion and bone-resorption are promoted by reactive oxygen species (ROS) signaling and by an unconventional low molecular weight species of La protein, located at the osteoclast surface. Here, we develop the hypothesis that La’s unique regulatory role in osteoclast multinucleation and function is controlled by a ROS switch in La trafficking. Using antibodies that recognize reduced or oxidized species of La, we find that differentiating osteoclasts enrich an oxidized species of La at the cell surface, which is distinct from the reduced La species conventionally localized within cell nuclei. ROS signaling triggers the shift from reduced to oxidized La species, its dephosphorylation and delivery to the surface of osteoclasts, where La promotes multinucleation and resorptive activity. Moreover, intracellular ROS signaling in differentiating osteoclasts oxidizes critical cysteine residues in the C-terminal half of La, producing this unconventional La species that promotes osteoclast fusion. Our findings suggest that redox signaling induces changes in the location and function of La and may represent a promising target for novel skeletal therapies.
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eLife assessment
This manuscript provides an important advance in our understanding of the molecular events that promote osteoclast fusion. Compelling data support the conclusion that an oxidized form of the ubiquitous protein La promotes osteoclast fusion following enrichment at the cell surface of osteoclast progenitors. These data improve our understanding of the processes that regulate bone resorption and will be of broad interest to researchers in the fields of cell biology and musculoskeletal physiology.
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Reviewer #1 (Public Review):
In this manuscript, Leikina et al. investigate the role of redox changes in the ubiquitous protein La in the promotion of osteoclast fusion. In a recently published manuscript, the investigators found that osteoclast multinucleation and resorptive activity are regulated by a de-phosphorylated and proteolytically cleaved form of the La protein that is present on the cell surface of differentiating osteoclasts. In the present work, the authors build upon these findings to determine the physiologic signals that regulate La trafficking to the cell membrane and ultimately, the ability of this protein to promote fusion. Building upon other published studies that show (1) that intracellular redox signaling can elicit changes in the confirmation and localization of La, and (2) that osteoclast formation is dependent …
Reviewer #1 (Public Review):
In this manuscript, Leikina et al. investigate the role of redox changes in the ubiquitous protein La in the promotion of osteoclast fusion. In a recently published manuscript, the investigators found that osteoclast multinucleation and resorptive activity are regulated by a de-phosphorylated and proteolytically cleaved form of the La protein that is present on the cell surface of differentiating osteoclasts. In the present work, the authors build upon these findings to determine the physiologic signals that regulate La trafficking to the cell membrane and ultimately, the ability of this protein to promote fusion. Building upon other published studies that show (1) that intracellular redox signaling can elicit changes in the confirmation and localization of La, and (2) that osteoclast formation is dependent on ROS signaling, the authors hypothesize that oxidation of La in response to intracellular ROS underlies the re-localization of La to the cell membrane and that this is necessary for its pro-fusion activity. The authors test this hypothesis in a rigorous manner using antioxidant treatments, recombinant La protein, and modification of cysteine residues predicted to be key sites of oxidation. Osteoclast fusion is then monitored in each condition using fluorescence microscopy. These data strongly support the conclusion that oxidized La is de-phosphorylated, increases in abundance at the cell surface of differentiating osteoclasts, and promotes cell-cell fusion. A strength of this manuscript is the use of multiple complementary approaches to test the hypothesis, especially the use of Cys mutant forms of La to directly tie the observed phenotypes to changes in residues that are key targets for oxidation. The manuscript is also well-written and describes a clearly articulated hypothesis based on a precise summation of the existing literature. The findings of this manuscript will be of interest to researchers in the field of bone biology, but also more generally to cell biologists. The data in this manuscript may also lead to future studies that target La for bone diseases in which there is increased osteoclast activity. The weaknesses of the manuscript are minor and predominantly relate to data presentation choices. These weaknesses do not detract from the overall conclusions of the study.
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Reviewer #2 (Public Review):
Summary:
Bone resorption by osteoclasts plays an important role in bone modeling and homeostasis. The multinucleated mature osteoclasts have higher bone-resorbing capacity than their mononuclear precursors. The previous work by authors has identified that increased cell-surface level of La protein promotes the fusion of mononuclear osteoclast precursor cells to form fully active multinucleated osteoclasts. In the present study, the authors further provided convincing data obtained from cellular and biochemical experiments to demonstrate that the nuclear-localized La protein where it regulates RNA metabolism was oxidized by redox signaling during osteoclast differentiation and the modified La protein was translocated to the osteoclast surface where it associated with other proteins and phospholipids to …
Reviewer #2 (Public Review):
Summary:
Bone resorption by osteoclasts plays an important role in bone modeling and homeostasis. The multinucleated mature osteoclasts have higher bone-resorbing capacity than their mononuclear precursors. The previous work by authors has identified that increased cell-surface level of La protein promotes the fusion of mononuclear osteoclast precursor cells to form fully active multinucleated osteoclasts. In the present study, the authors further provided convincing data obtained from cellular and biochemical experiments to demonstrate that the nuclear-localized La protein where it regulates RNA metabolism was oxidized by redox signaling during osteoclast differentiation and the modified La protein was translocated to the osteoclast surface where it associated with other proteins and phospholipids to trigger cell-cell fusion process. The work provides novel mechanistic insights into osteoclast biology and provides a potential therapeutic target to suppress excessive bone resorption in metabolic bone diseases such as osteoporosis and arthritis.
Strengths:
Increased intracellular ROS induced by osteoclast differentiation cytokine RANKL has been widely studied in enhancing RANKL signaling during osteoclast differentiation. The work provides novel evidence that redox signaling can post-translationally modify proteins to alter the translocation and functions of critical regulators in the late stage of osteoclastogenesis. The results and conclusions are mostly supported by the convincing cellular and biochemical assays,
Weaknesses:
The lack of in vivo studies in animal models of bone diseases such as postmenopausal osteoporosis, inflammatory arthritis, and osteoarthritis reduces the translational potential of this work.
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