Hem1 promotes osteoclast fusion and bone resorption in mice

Hem1 (Hematopoietic protein 1), a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins, is essential for lymphopoiesis and innate immunity and for the transition of hematopoiesis from the fetal liver to the bone marrow. However, the role of Hem1 in bone cell differentiation and bone remodeling is unknown. Here, we show that deletion of Hem1 resulted in a markedly increase in bone mass due to defective bone resorption in mice of both sexes. Hem1-deficient osteoclast progenitors were able to differentiate into osteoclasts, but the osteoclasts exhibited impaired osteoclast fusion and decreased bone-resorption activity, potentially due to cytoskeletal disorganization and decreased mitochondrial respiration. Transplantation of bone marrow hematopoietic stem and progenitor cells from wild-type into KO mice increased bone resorption and normalized bone mass. These findings indicate that Hem1 plays a pivotal role in the maintenance of normal bone mass.


Introduction
Bone is a highly dynamic tissue that responds and adapts to changes in systemic signals and to mechanical forces. Bones regenerate periodically in discrete sites via a remodeling process through which old or damaged bone is resorbed by osteoclasts and is replaced with new bone by osteoblasts 1,2 .
Osteoclasts differentiate from hematopoietic precursor cells of the monocyte/macrophage lineage in response to two critical osteoclastogenic cytokinesmacrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-B ligand (RANKL) 3 . Osteoclasts develop in several steps, beginning with proliferation of hematopoietic progenitor cells that differentiate to mononuclear preosteoclasts that then fuse to become multi-nucleated mature osteoclasts 3 . These cells are uniquely capable of dissolving and digesting the bone matrix due to their ability to trigger actin polymerization in actin ring and to form a "podosome belt" that tightly adheres to the bone area that is targeted for removal, creating a sealed microenvironment into which the cells secrete protons and lysosomal enzymes 3,4 . The high energy demands of these tasks are likely the reason for the abundance of mitochondria within osteoclasts, a distinct cellular feature of these cells 5,6 . The balancing act of healthy bone remodeling is accomplished by osteoblasts refilling each resorption cavity with new bone. Osteoblasts derive from mesenchymal stem cells present in the bone marrow 1 . Osteoblast differentiation occurs in response to osteoblastformation signals, including matrix-derived factors released during bone resorption.
The contribution of osteoclast-derived signals or factors released during resorption to osteoblast generation is referred to as "coupling" 7,8 .
Hematopoietic protein 1 (Hem1), also known as Nck-associated protein 1-like (NAP1l or Nckap1l), is a member of the Hem family of cytoplasmic adaptor proteins.
Orthologues of Hem1 in lower organisms, such as Drosophila melanogaster and Caenorhabditis elegans, are essential for cytoskeletal reorganization, embryonic cell migration, and morphogenesis [9][10][11] . Studies in mice and in cell lines indicate that Hem1 is a component of the ''WAVE (WASP [Wiskott-Aldrich syndrome protein]family verprolin homologous protein)'' complex, which signals downstream of activated Rac and upstream of the Arp2/3 complex to stimulate actin polymerization in response to immunoreceptor signaling 9,12 . Recent work from our group has shown that mice with deletion of Hem1 exhibit growth retardation and premature death at about 6 weeks. These defects were associated with premature exhaustion of neonatal bone marrow hematopoietic stem cells, indicating that Hem1 is required for hematopoiesis to transition from the fetal liver to the bone marrow 13 . In contrast, Hem-1 is dispensible for the normal development and function of the other organ systems. Because osteoclasts differentiate from the hematopoietic lineage, we examined the roles played by Hem1 in the skeleton. Here, we show that deletion of Hem1 led to a defect in osteoclast maturation, a decrease in bone-resorption and a marked increase in trabecular bone volume. All these abnormalities could be corrected by transplantation of normal bone marrow hematopoietic stem and progenitor cells.

Hem1 knockout mice have increased bone mass.
To address the role of Hem1 in bone remodeling, we generated Hem1 knockout mice as described previously 13 . At 5.5 weeks of age, Hem1 knockout mice were smaller than their littermate wild-type controls, as evidenced by reduced body size, body weight, and femoral length ( Fig. 1a and Supplementary Fig. 1), but the morphology of the femoral growth plate was not affected (Fig. 1b). Consistent with the reduced femoral length in Hem1 knockout mice, the femurs had lower cortical thickness, total area, and medullary area than wild-type littermates ( Fig. 1c and  Hem1 knockout mice have more osteoclasts but less bone resorption than wildtype mice. To determine the cellular basis of the increase in trabecular bone mass, we conducted histological analyses to enumerate osteoblasts and osteoclasts in the distal femur. Consistent with the micro-CT results, trabecular bone volume and trabecular number were greater but trabecular spacing was smaller in Hem1 knockout mice than in wild-type controls (Fig. 2a). Surprisingly, the knockout mice had increased numbers of osteoclasts, as determined by tartrate-resistant acid phosphatase (TRAP) staining in histological sections of the femur (Fig. 2b). These changes were associated with an increase in the number of myeloid cells in the bone marrow ( Supplementary Fig. 3).
However, bone resorption as determined by serum levels of CTX was severely reduced from that seen in wild-type controls (Fig. 2d).
Consistent with the observed reduction in bone resorption, osteoblast numbers were lower in Hem1 knockout mice (Fig. 2c). To establish whether the number of mesenchymal progenitors was affected by Hem1 deletion, we quantified the number of CFU-fibroblasts (CFU-F), CFU-osteoblasts (CFU-OB), and CFU-adipocytes (CFU-A) formed by mesenchymal progenitors from wild-type and Hem1 knockout mice in ex vivo bone marrow cultures. The numbers of CFU-F, CFU-OB, and CFU-A were greater in Hem1 knockout mice than in WT mice ( Fig. 2e-g). These results indicate that the low osteoblast number in knockout mice are not due to reduced mesenchymal progenitors. Taken together, these findings suggest that the high bone mass of Hem1 knockout mice is due to a decrease in osteoclastic bone resorption.

Deletion of Hem1 decreases actin ring formation and osteoclast fusion in vitro.
To investigate the role of Hem1 in osteoclastogenesis, bone marrow macrophages were cultured in the presence of M-CSF and RANKL. The Hem1 mRNA expression and protein levels was about 3-fold higher in mature osteoblast than macrophages or preosteoclasts from wild-type mice. (Fig. 3a-b). Macrophages from Hem1 knockout mice ( Fig. 3c) failed to form multinucleated giant osteoclasts (with more than 10 nuclei) but formed a higher number of smaller osteoclasts (with 3-5 nuclei) than those from wild-type mice (Fig. 3d). Levels of mRNA for markers of mature osteoclast differentiation (TRAP, Cathepsin K, Calcitonin receptor, Oscar) were significantly lower in cultures from Hem1-deficient mice than in those from wild-type littermates (Fig. 3e). In contrast, expression of NFATc1 and c-Fos, two essential transcription factors for early osteoclast differentiation, were not affected by Hem1 deletion (Fig. 3f), suggesting that Hem1 does not affect the early stage of osteoclast differentiation.
To determine whether osteoclasts from Hem1-deficient mice could resorb bone, we cultured osteoclasts from wild-type and Hem1 knockout mice on OsteoAssay plates and found that the area resorbed ( Fig. 3g) and actin ring formation ( To confirm that the defects seen in bone resorption of Hem1 knockout mice were due to direct effects of Hem1 in macrophages, we expressed Hem1 in bone marrow macrophage cultures from Hem1 knockout mice (Fig. 4a). Expression of Hem1 increased osteoclast formation (Fig. 4b) and resorption capacity by 2 to 3-fold (Fig. 4c).
Cells of the mesenchymal lineage including stromal cells are major producers of RANKL and other cytokines that support osteoclastogenesis. We also examined whether osteoclast differentiation in Hem1 knockout mice could be affected indirectly by bone marrow-mesenchymal cells. To this end, we co-cultured bone marrow macrophages from wild-type mice with stromal cells from wild-type or Hem1 knockout mice. Stromal cells from either genotype equally supported osteoclast formation ( Supplementary Fig. 4). These data further support to the idea that Hem1 contributes directly to the formation of multinucleated osteoclasts. Hem1 knockout mice with those from wild-type mice. In osteoclasts from Hem1 knockout mice, mitochondrial respiration was significantly diminished (Fig. 5a), and ATP-linked respiration, proton leakage, maximum respiration (the OCR when electron transport chain operates at maximum capacity), reserve respiratory capacity (a cell's flexibility to increase oxygen consumption during increased energy demands), and nonmitochondrial respiration were reduced ( Fig. 5b-f). Consistent with these inhibitory effects of Hem1 deletion on respiration, osteoclasts from Hem1 knockout mice had decreased ATP production in response to RANKL stimulation (Fig. 5g).
Our recent work indicates that inactivation of c-Abl, a tyrosine kinase that regulates actin polymerization and cell migration [18][19][20][21] , contributes to the impairment of hematopoietic stem cells in Hem1-deficient mice 13 . Here, we examined whether Hem1 activates osteoclast formation via c-Abl signaling. Compared to osteoclasts from wildtype mice, those from Hem1 knockout mice had less phosphorylation of the c-Abl downstream target Crkl but no changes in total protein levels of Crkl (Fig. 5h). Addition of imatinib, a synthetic small-molecule inhibitor of c-Abl, to cultures of osteoclast progenitors from wild-type C57BL/6 mice resulted in decreased formation of osteoclasts (Fig. 5i), and most of these osteoclasts were small (Fig. 5i), reminiscent of osteoclasts from Hem1 knockout mice.

Skeletal phenotype of Hem1 knockout mice is rescued by transplantation of bone marrow hematopoietic stem and progenitor cells from wild-type mice.
Our previous studies showed that Hem1 knockout mice exhibited significant reductions in the numbers of total bone marrow nucleated cells, hematopoietic progenitor cells, hematopoietic stem cells, and cobblestone area forming cells, which could be corrected by transplantation of normal wild-type bone marrow hematopoietic stem and progenitor cells 13 . Therefore, we investigated whether transferring these cells could rescue the bone effects due to Hem1 deletion. We transplanted 1x10 6 Lin -CD45 + bone marrow cells from normal C57BL/6 congenic (CD45.1/GFP) mice into 3-week-old non-ablated CD45.2 wild-type and Hem1 knockout mice (Fig. 6a). Ten weeks after transplantation, most components of the hematopoietic system, including hematopoietic stem and progenitor cells, were comparable in wild-type and Hem1 knockout recipient mice (Supplementary Figure 5). In Hem1 knockout recipient mice, almost 90% of cells in the peripheral blood and bone marrow were derived from CD45.1/GFP donor cells; however, in wild-type recipient mice, no donor cells were detected. These results indicate that the transplanted normal hematopoietic cells reconstituted the hematopoietic system in Hem1 knockout recipient mice. Transplantation with normal hematopoietic cells also restored body size (Fig. 6b). Although not fully rescued, femoral length (Fig. 6c), cortical thickness, total area, and medullary area were much improved ( Fig. 6d and e) in transplanted Hem1 knockout mice than in non-transplanted Hem1 knockout mice ( Fig. 1c and d). Remarkably, trabecular bone volume and microarchitecture in the distal femur ( Fig. 6f and g) and serum levels of CTx in Hem1 knockout recipient mice were similar to wild-type (Fig. 6h). Consistently, in vitro boneresorption ability was comparable for osteoclasts from both genotypes of recipient mice ( Fig. 6i). In addition, osteoclast cultures from the bone marrow of transplanted Hem1 knockout mice had significantly increased osteoclast formation (Fig. 6j) and upregulated the expression of TRAP, Cathepsin K, Calcitonin receptor, and Oscar ( Fig.   6k). Actin ring formation in osteoclasts from transplanted Hem1 knockout mice was comparable to that in wild-type mice, as was expression of Dc-stamp, Oc-stamp, Atp6v0d2, and Nfatc1 ( Fig. 6l and m).

Discussion
The pathways of osteoclastogenesis that regulate RANKL-induced osteoclast differentiation have been studied extensively, but less is known about osteoclast fusion, a unique feature of osteoclast maturation that is required for the proper bone resorption.
Herein, we provide evidence that the hematopoietic-specific WAVE complex scaffold Hem1 is required for osteoclast fusion and bone-resorption activity and, thereby, A defect in hematopoiesis contributes to the osteopetrosis with abnormal bone remodeling 29,30 . Transplantation of bone marrow hematopoietic stem and progenitor cells markedly improves severe osteopetrosis associated with the defect in bone marrow and offers the best chance of longer-term survival for individuals with this type of disease 31,32 . Here, we showed that transplanting normal hematopoietic stem and progenitor cells into Hem1 knockout mice fully restored cancellous bone volume and microarchitecture in the distal femur, as well as osteoclast fusion and bone-resorption activity. More importantly, the transplantation also rescued growth retardation and premature death of Hem1 knockout mice. Collectively, these findings suggest that Hem1 is essential during growth for bone remodeling and for hematopoietic stem cell maintenance.
In line with our findings, studies of short interfering RNA oligonucleotides in an osteoclast cell line revealed that the Arp2/3 complex regulates actin polymerization and promotes bone resorption 33 , and other studies showed that the Arp2/3 complex locates in the core of the podosomes that make up actin rings, where it co-localizes with cortactin, a known regulator of Arp2/3 complex activity 34,35 . In yeast, the Arp2/3 complex promotes actin polymerization by regulating mitochondrial dynamics, but little is known about its role in mitochondria metabolism in mammalian cells 16,17 . Here, we showed that in osteoclasts deletion of Hem1 decreased mitochondrial respiration.
Overall, these findings support the notion that, Hem1, a component of the Rac-WAVE-Arp2/3 pathway, exerts effects in osteoclasts at least in part via an effect on mitochondrial function. Support for a link between Hem1, mitochondria, and bone resorption is provided by studies in which mitochondria activity has been attenuated in osteoclasts [36][37][38] . These studies indicate that normal mitochondria metabolism is critical for osteoclast fusion and bone resorption. Nonetheless, future work is required to identify the range of target proteins and mitochondrial pathways that are responsible for the effects of Hem1 on osteoclast development.
A decrease in bone resorption was, most likely, responsible for the increase in trabecular bone mass in Hem1 knockout mice. Nonetheless, changes in mesenchymal lineage cell number, specifically CFU-F, CFU-Ad, CFU-Ob, and osteoblast were also noted. These changes are, most likely, indirect as the expression of Hem1 is very low in mesenchymal lineage cells (CD45 -) from wild-type mice ( Supplementary Fig. 6).
Strikingly, Hem1 knockout mice exhibited low numbers of osteoblast on trabecular bone surfaces, which could not be explained by changes in progenitor cell numbers indicated by CFU assays. One possible explanation for the decrease in osteoblasts could be the decrease in growth factors released from the matrix during bone resorption 7,8 . In Cellular bioenergetics. Application of extracellular flux analysis was performed as we previously described 45,46 . Bone marrow macrophages were plated and treated with 30 ng ml -1 M-CSF and 30 ng ml -1 RANKL for 3 days. The media in the wells was replaced with XF assay media and the plate was kept in a non-CO2 incubator for 20 minutes at 37 °C. After recording three total cellular respiration measurements with the XF96 analyzer, 10 µg ml -1 oligomycin was added to inhibit mitochondrial ATP synthase and measure the decrease in the oxygen-consumption rate that is linked to ATP turnover.
To determine the maximal respiration potential of the cells, 10 µM FCCP (an uncoupler of oxidative phosphorylation) was used. The amount of non-mitochondrial oxygen consumption was determined by inhibiting the respiratory chain activity with an antimycin A and 10 µM rotenone cocktail. These data were used to calculate the mitochondrial basal respiration, ATP-linked respiration, reserve respiratory capacity, and proton leak. GraphPad Software, after determining that the data were normally distributed and exhibited equivalent variances. In the event that ANOVA justified post hoc comparisons between group means, these were conducted with Tukey's multiplecomparisons test.

Hematopoietic cell transplantation. Bone marrow mononuclear cells were isolated
Data availability. The data that support the findings of this study are available from the corresponding author upon reasonable request.   Lines and error bars represent mean ± SD. P values determined using Student's t-test.

Supplementary Files
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