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Human Molecular Genetics Advance Access originally published online on December 18, 2007
Human Molecular Genetics 2008 17(7):936-948; doi:10.1093/hmg/ddm366
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Rac1 mediates the osteoclast gains-in-function induced by haploinsufficiency of Nf1

Jincheng Yan1,2,5,{dagger}, Shi Chen1,2,{dagger}, Yingze Zhang5, Xiaohong Li1,2, Yan Li1,2, Xiaohua Wu1,2, Jin Yuan1,2, Alexander G. Robling3, Reuben Karpur1,2,4, Rebecca J. Chan1,2 and Feng-Chun Yang1,2,*

1 Department of Pediatrics 2 Herman B Wells Center for Pediatric Research 3 Department of Anatomy and Cell Biology and 4 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA 5 Department of orthopedic, The 3rd Hospital, Hebei Medical University, Shijiazhuang, China

* To whom correspondence should be addressed at: Indiana University School of Medicine, Cancer Research Institute, 1044 W. Walnut St., R4/427, Indianapolis, IN 46202, USA. Tel: +1 3172744178; Fax: +1 3172748679; Email: fyang{at}iupui.edu

Received October 30, 2007; Revised November 30, 2007; Accepted December 5, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Neurofibromatosis type I (NF1) is a congenital disorder resulting from loss-of-function of the tumor suppressor gene, NF1, a GTPase-activating protein for p21ras. Fifty percent of NF1 patients have osseous manifestations including a high incidence of osteoporosis. Osteoclasts are specialized macrophage/monocyte lineage-derived cells that resorb bone and NF1 haploinsufficient osteoclasts have abnormal Ras-dependent bone resorption. Ras-regulated functions are in part mediated via the activation of small Rho family of GTPases including the Rac-GTPases. In the present study, we demonstrate that the Rho-GTPase Rac1 is a crucial Ras-mediated effector in Nf1 haploinsufficient (+/–) osteoclasts. Nf1+/– mice were intercrossed with conditional Rac1flox/floxMxcre+ (Rac1–/–) mice to generate Nf1+/–; Rac1–/– mice. Genetic disruption of Rac1 restored the pathological increase in osteoclast progenitor cells in Nf1+/– mice and was sufficient to correct the increased Nf1+/– osteoclast motility and osteoclast belt formation, an f-actin structure observed in mature osteoclasts critical for bone resorption and lytic activity. Finally, we demonstrate that Nf1+/–; Rac1–/– osteoclasts have normalized Erk activation compared with Nf1+/– osteoclasts, a biochemical function critical for osteoclast formation, actin organization and motility. Collectively, these data demonstrate that Rac1 critically contributes to increased osteoclast function induced by haploinsufficiency of Nf1 and implicate Rac1 as a rational therapeutic target for osteoporosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Neurofibromatosis type 1 (NF1) is a common, autosomal-dominant disorder caused by mutations in the NF1 tumor suppressor gene (1–3). It is a member of a group of disorders including NF1, Noonan syndrome, cardio-facio-cutaneous syndrome, Costello syndrome and LEOPARD syndrome, all of which commonly bear germline mutations within key genes contributing to the Ras-Raf-Mek-Erk signaling cascade, collectively designated neuro-cardio-facio-cutaneous syndromes (4). Although individuals with NF1 have a higher incidence of malignancies than general population, both clinical data from NF1 patients and experimental studies in Nf1 hyterozygous (+/–) mice demonstrate that haploinsufficiency of NF1 (Nf1) also results in multiple non-malignant phenotypes (5–7). For example, Nf1+/– mice have deficits in long term learning, similar to the spatial–visual dis-coordination observed in NF1 patients (8,9). The high frequency of other non-malignant manifestations in NF1 patients, including cerebrovascular disease (10), renal vascular hypertension (11–13) and osseous abnormalities (14–22) including osteoporosis, implies the importance of NF1 haploinsufficiency in multiple lineages. One noteworthy complication suffered by NF1 patients, as well as patients afflicted with neuro-cardio-facio-cutaneous syndromes, in general, is skeletal abnormalities (15,16,18,21,23–30). Several groups have established that NF1 patients have reduced bone mineral density, suggesting that these patients are at high risk for osteoporosis (14,16,18,20,21,31–33). We recently reported that osteoclasts derived from NF1 patients and Nf1+/– mice have elevated migration, adhesion and bone resorption, providing a cellular mechanism underlying these clinical observations (34). Previous studies demonstrated that osteoclast differentiation and function depend on the Ras-phospho-inositol-3-kinase (PI3K)-Erk signaling cascade (35–39) and, consistently, our studies indicate that the gain-of-function of the Nf1+/– osteoclasts is, at least in part, caused by hyperactivation of macrophage colony-stimulating factor (M-CSF)-stimulated Ras, Akt and Erk (34). The central role of Ras in mediating Nf1+/– osteoclast hyperactivation has been validated by normalization upon ectopic expression of the Nf1 GTPase-activating-protein-related domain (GRD); however, the specific Ras effector molecules contributing to the gain-of-function within the osteoclast compartment are unknown.

Bone remodeling is a complex, highly orchestrated process that is mediated by osteoclasts which function by resorbing the bone while migrating along the bone surface. Osteoclasts are hematopoietic stem cell (HSC)-derived bone-resorbing cells that arise from monocyte/macrophage precursors by cell fusion and differentiation. Two cytokines necessary and sufficient for osteoclastogenesis, receptor activator of nuclear factor kappaB ligand (RANKL) and M-CSF, are both produced by mesenchymal cells in the bone marrow environment (40). Following maturation, osteoclasts home and adhere to the bone surface via {alpha}vβ3 integrin and then polarize and secrete hydrochloric acid and acidic proteases which degrade the bone extracellular matrix (41,42). During osteoclast differentiation, progression of podosome organization is characterized by cluster, ring and belt development (43), with the podosome belt found in the most mature osteoclasts. Mature osteoclasts are the only cell type that contains podosomes that arrange into a precisely defined circle at the cell periphery.

Downstream effectors of hyperactivated Ras, such as the Rho GTPases, likely play a role in the observed gain-of-function in Nf1+/– osteoclasts and potentially contribute to additional dysplastic and erosive bone diseases including osteoporosis, Paget's disease of bone, bone metastases and wear particle-induced osteolysis following arthroplasty (44–47). Rho GTPases function downstream of Ras and act as binary switches, cycling between an inactive (GDP-bound) and active (GTP-bound) state, to regulate osteoclast actin ring formation, bone resorption and development of filamentous actin structures associated with migration and adhesion (48,49). Rac1, a ubiquitously expressed Rho GTPase, and Vav3, a Rho guanine exchange factor which promotes the conversion of inactive Rac-GDP to active Rac-GTP, are essential in the regulation of osteoclast function (50–53). Multiple lines of evidence indicate that several Rac-related cellular functions depend on PI3K activity and that products of PI3K activity activate Vav (49,54–57). Recent studies demonstrate that Rac1 plays a more prominent role in osteoclast development and function than that of Rac2 both in vitro and in vivo (58). Based on these studies, as well as previous studies conducted in our laboratory demonstrating that the gain-of-function in Nf1+/– mast cells, also of myeloid origin, is corrected by genetic deletion of Rac2 (55,59,60), we were interested to determine if Rac1, Rac2 or both Rac1 and Rac2 play a crucial role in the increased osteoclast functions observed in Nf1+/– mice and NF1 patients. To examine this question, we used genetic and biochemical studies to determine whether hyperactivation of Rac1 or Rac2 contribute to the osteoclast gain-of-function observed in the Nf1+/– osteoclasts. We report that the Rho GTPase Rac1, but not Rac2, contributes to M-CSF-induced hyperactivation of osteoclast functions, including osteoclast formation, migration and bone resorption, in Nf1+/– mice. Furthermore, we show that Rac1 mediates signals emanating from the Ras-Erk signaling cascade and that inhibition of this signaling pathway is critical for normalization of the osteoclast gain-of-function induced by Nf1 haploinsufficiency. Collectively, our results suggest that Rac1 is a potential molecular target for the treatment of dysplastic and erosive bone diseases.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Nf1+/– osteoclast progenitors have elevated Rac1-GTPase activation
We previously reported that Nf1+/– mice have increased osteoclast differentiation and actin-mediated functions associated with increased M-CSF-stimulated Ras, Akt and Erk activation (34). Rac1-GTPase has been previously shown to be activated during osteoclast migration and differentiation (51). In order to test the hypothesis that hyperactivation of Rac-GTPase in Nf1+/– mice are required for these pathological functions, we intercrossed Nf1+/– mice with Rac1flox/flox; Mxcre+ mice that disrupts the Rac1 transgene in hematopoietic cells. To induce genetic deletion of Rac1, mice were treated with poly I:C as described in the Materials and Methods. Genetic disruption of the Rac1 gene was verified by PCR of the recombinant Rac1 gene (data not shown) and by demonstrating that the Rac1 protein was absent as detected by western blotting for total Rac1 (Fig. 1A). For simplicity, mice containing the disrupted Rac1flox/flox allele will be referred to as Nf1+/–; Rac1–/– and Rac1–/–.


Figure 1
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  		Figure 1. Rac1 GTPase is hyperactivated in Nf1+/– osteoclasts. (A) Rac1 total protein and Rac1 GTPase activation was examined at baseline and 1 min following M-CSF stimulation in murine osteoclast progenitors. (B) Rac2 GTPase activation was examined at baseline and 1 min following M-CSF stimulation in murine osteoclast progenitors. Each western blot evaluation was performed on three independent occasions.

 
To examine Rac1- and Rac2-GTPase activation, we compared M-CSF-stimulated Rac1 and Rac2 GTPase activity in Nf1+/– osteoclast progenitors to that of WT osteoclast progenitors using a Pak-pulldown assay. Following stimulation with M-CSF, both Rac1- and Rac2-GTPase are activated in WT and Nf1+/– osteoclast progenitors (Fig. 1A and B). However, Nf1+/– cells demonstrate higher basal and M-CSF-stimulated Rac1-GTPase activation when compared with that in WT cells (Fig. 1A). We also noticed a higher level of total Rac1 protein in Nf1+/– cells when compared with WT cells while there is a similar level of β-actin expression (Fig. 1A). As expected, Rac1-GTPase was not detected in Rac1–/– cells or Nf1+/–; Rac1–/– cells (Fig. 1A). Interestingly, basal level of Rac2-GTPase activation was also increased in Nf1+/– cells when compared with that in WT cells (Fig. 1B). Constitutively hyperactivated Rac2 in the Rac1–/– and Nf1+/–; Rac1–/– cells may be due to compensation secondary to the absence of Rac1. Collectively, these biochemical studies indicate that Rac1-GTPase activity is selectively hyperactivated in Nf1+/– osteoclasts.

Genetic disruption of Rac1 corrects osteoclast progenitor colonies and osteoclast formation derived from Nf1+/– mice
We previously found that Nf1+/– mice have a 2-fold increase in the number of osteoclast progenitor cells compared with syngeneic WT controls (34). To test the functional significance of Rac1-GTPase in Nf1 induced osteoclastogenesis, we utilized clonogenic assays to measure colony-forming-unit-macrophage (CFU-M) and osteoclast progenitors following tartrate resistant acid phosphatase (TRAP) staining (TRAP+ CFU-M) from freshly isolated bone marrow cells. As reported previously, Nf1+/– mice have increased CFU-M and TRAP+ CFU-M progenitors compared with that in WT mice (Fig. 2A and B). Further, Rac1–/– mice have significantly lower numbers of these progenitors than WT mice. Importantly, genetic deletion of Rac1 normalized the increased number of CFU-M in Nf1+/–; Rac1–/– mice to WT levels (Fig. 2A and B). Finally, in contrast to the results obtained upon genetic deletion of Rac1, genetic deletion of Rac2 did not reduce total numbers of CFU-M or TRAP+ CFU-M compared with WT and did not correct the increased number of TRAP+ CFU-M or CFU-M in the Nf1+/–; Rac2–/– mice (data not shown).


Figure 2
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  		Figure 2. Genetic deletion of Rac1 normalizes osteoclast progenitor development. (A) BMMNCs of the indicated genotypes were cultured in agar-based media containing M-CSF 20 ng/ml and RANKL 50 ng/ml for 7 days and CFU-M were quantitated based on morphology. Experiment conducted on four independent occasions. (B) To examine TRAP+ CFU-M, colonies were fixed in citrate/acetone/formaldehye solution and histochemically stained for TRAP activity to detect osteoclast progenitors. Experiment conducted on four independent occasions. (C) Representative photomicrograph of osteoclast progenitors of the indicated genotypes generated in vitro following culture in {alpha}-MEM, 10% FBS, RANKL 50 ng/ml and M-CSF 30 ng/ml. Osteoclast progenitors were identified by staining for TRAP activity. (D) Quantitative analysis of osteoclast progenitors following in vitro culture. Experiment conducted on five independent occasions. (E) Osteoclast progenitors were pulsed with [3H] thymidine growth factors for 6 h. Experiment performed on three independent occasions.

 
To further evaluate the role of Rac1 in Nf1+/– osteoclast development, we next established liquid cultures to evaluate osteoclast formation in vitro followed by TRAP staining. Consistent with the progenitor data, Nf1+/– bone marrow cells have significantly increased osteoclast formation when compared with all other experimental groups as shown qualitatively in Figure 2C and quantitatively in Figure 2D. In addition, Rac1–/– bone marrow cells generated the lowest numbers of osteoclasts of all genotypes while Rac2–/– bone marrow cells yielded osteoclasts that were comparable in number to WT bone marrow (data not shown). Furthermore, genetic disruption of Rac1 reduced the osteoclast formation to normal levels in Nf1+/– culture, while Rac2–/– bone marrow cells have identical level of osteoclast formation as WT culture (data not shown). Based on thymidine incorporation assays, reduced proliferation, at least in part, contributes to the reduced number of CFU-M, TRAP+ CFU-M and in vitro osteoclast formation derived from the Rac1–/– mice (Fig. 2E). Taken together, these data indicate that Rac1 plays a vital role in regulating osteoclast differentiation and development. The central function of Rac1-GTPase, in contrast to that of Rac2-GTPase, is highlighted by the finding that the enhanced osteoclastogenesis induced by Nf1 haploinsufficiency is functionally corrected by genetic deletion of Rac1, in spite of the presence of constitutively activated Rac2-GTPase activity observed in these cells.

Genetic deletion of Rac1 corrects the gain-of-function of Nf1+/– osteoclast migration
The ability to migrate across the bone surface is a key cellular function required for bone resorption by osteoclasts. To evaluate whether gain-of-migration observed in Nf1+/– cells is corrected by genetic disruption of Rac1, transwell migration assays were performed utilizing purified populations of TRAP+ cells. A representative photomicrograph of the migrating TRAP+ cells is shown in Figure 3A. Nf1+/– osteoclasts have a 2-fold increase in migration when compared with WT cells shown qualitatively in Figure 3A and quantitatively in Figure 3B. Disruption of Rac1 in osteoclasts resulted in a significant reduction in the M-CSF-mediated migration of Nf1+/–; Rac1–/– cells to a migration level similar to that of WT osteoclasts (Fig. 3A and B) while genetic disruption of Rac2 did not normalize this gain in function (data not shown).


Figure 3
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  		Figure 3. Genetic deletion of Rac1 corrects the gain-of-function of Nf1+/– osteoclast migration and bone resorption. (A) Representative photomicrograph of the migrating TRAP+ osteoclast progenitors through a 8µm polycarbonate filter coated with vitronectin in response to {alpha}-MEM, 0.1% BSA and M-CSF (30 ng/ml). (B) Osteoclast migration quantitative analysis of the indicated genotypes. Experiment conducted on four independent occasions.

 
Rac1 regulates the actin organization in Nf1+/– osteoclasts
The osteoclast bone resorbing capacity is dependent on the organization of the actin cytoskeleton to form a specialized cell–extracellular matrix that initiates degradation of bone matrix by the controlled release of proteases (61). This complex structure is formed by the coalescence of multiple small actin-based adhesion structures called podosomes that organize into progressively larger patterns identified as clusters, rings and ultimately belts that form a functional sealing zone. Given the role of Rac-GTPases in other cytoskeletal functions in myeloid cells (55,62,63), we evaluated the impact of Rac1 on modulating the actin cytoskeleton and scored the podosome organization using established criteria (43). Nf1+/– cultures demonstrated markedly larger multinucleated osteoclasts compared with that in WT cultures following the phalloidin staining as observed in the low power field (LPF) (10x, Fig. 4A) and a higher power field (40x, Fig. 4B). The red arrowheads in Figure 4B indicate representative belt formation in the osteoclasts while white and yellow colored arrows show clusters and rings, respectively. Nf1+/– osteoclasts demonstrated significantly higher levels of belt formation as compared with all other genotypes while Rac1–/– osteoclasts demonstrated significantly less numbers of both belt and ring structures (Fig. 4C). Upon genetic deletion of Rac1, the Nf1+/–; Rac1–/– osteoclasts demonstrated belt formation at a similar level to that of WT osteoclasts (Fig. 4D). These data indicate that Rac1 plays an essential role in functional f-actin organization and suggest that inhibition of Rac1 in the setting of Nf1 haploinsufficiency is able to normalize osteoclast hyperactivity by correcting the cytoskeletal organization of f-actin-based structures.


Figure 4
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  		Figure 4. Rac1 regulates the actin organization in Nf1+/– osteoclasts. (A and B) Representative photomicrograph of osteoclast progenitors following staining with FITC-conjugated phalloidin at 10x and 40x magnification, respectively. Arrows in (B) indicate the belt forming cells. (C) Classification of podosome organization within osteoclast progenitors of the indicated genotypes was quantitated morphologically into three categories: cluster, ring and belt. Experiment conducted on four independent occasions. (D) Quantitative analysis of belt formation in each of the indicated genotypes. Experiment conducted on four independent occasions.

 
The increased lytic activity of Nf1+/– osteoclasts is mediated by Rac1
To functionally assess whether the increase in belt formation is associated with an increase in skeletal lytic activity, we next cultured osteoclasts on dentine slices and examined the number and area or ‘pits’ that are resorbed. A representative photomicrograph of bone resorption assay is shown in Figure 5A. The total area/LPF resorbed is shown quantitatively in Figure 5B. A 2-fold increase in the resorbed area was observed in Nf1+/– culture compared with the WT culture. Rac1–/– cells demonstrate only limited bone resorption. Pit formation was corrected to WT levels in Nf1+/–; Rac1–/– cells. Collectively, these data indicate that Rac1 is critical for the observed increased bone resorption performed by Nf1+/– osteoclasts and that inhibition of Rac1 function is able to normalize the increased osteoclast growth, differentiation and bone-resorbing capacity induced by haploinsufficiency of Nf1.


Figure 5
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  		Figure 5. Genetic deletion of Rac1 corrects the gain-of-function of Nf1+/– osteoclast bone resorption. (A) Representative photomicrograph of bone resorption assay following culture of osteoclast progenitors on dentine slices. The number and area of the resorbed regions, referred to as ‘pits’, was quantified. (B) Resorption quantitative analysis of the indicated genotypes. Experiment conducted on four independent occasions.

 
Hyperactivation of Erk in Nf1+/– osteoclast progenitors is normalized by deletion of Rac1 and inhibition of the Ras-Erk pathway corrects the aberrant cytoskeletal organization observed in Nf1+/– osteoclasts
The mitogen-activated protein kinases, Erk1 and Erk2, have been directly linked to podosome formation in multiple lineages (39,63,64). Given that Rac-GTPases have been linked by crosstalk from the PI3K to the Ras-MAPK pathway, we assessed whether the functional correction of osteoclast formation in Nf1+/–; Rac1–/– osteoclasts was linked to a reduction in biochemical activation of Erk as well as Akt phosphorylation, a sensitive measure of PI3K activity. As shown in Figure 6A, Erk1 and Erk2 were hyperactivated in Nf1+/– osteoclasts compared with WT in response to M-CSF stimulation and were only minimally activated in the Rac1–/– osteoclasts. Genetic disruption of Rac1 corrected the elevated M-CSF-stimulated levels of phospho-Erk1/2 in the Nf1+/–; Rac1–/– osteoclasts to levels observed in the WT cells. Similarly, increased Akt phosphorylation was also observed in Nf1+/– osteoclasts following M-CSF stimulation when compared with that in WT cells (Fig. 6B). Minimal M-CSF-stimulated Akt phosphorylation was observed in Rac1–/– osteoclast cells. Genetic deletion of Rac1 in Nf1+/– osteoclasts normalized M-CSF-stimulated Akt phosphorylation levels to that of WT. Together, these data suggest that signals from Rac1 to PI3K and Erk contribute to the cytoskeletal anomalies observed in the Nf1+/– osteoclasts leading to the gain-of-function in osteoclast differentiation, migration and bone resorption.


Figure 6
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  		Figure 6. Hyperactivation of MAPK in Nf1+/– osteoclast progenitors is normalized by deletion of Rac1 and inhibition of the MAPK pathway corrects the aberrant cytoskeletal organization observed in Nf1+/– osteoclasts. (A and B) Osteoclast progenitors of the indicated genotypes were serum and growth-factor deprived, stimulated with M-CSF for 2 and 5 min, and evaluated for phosphorylation of Erk and Akt. Western blots performed on three independent occasions using osteoclast progenitors derived from three independent experimental groups of mice. (C) Representative photomicrographs of osteoclast progenitors of the indicated genotypes following culture in {alpha}-MEM, 10% FBS, RANKL 50 ng/ml and M-CSF 30 ng/ml in the presence or absence of PD98059 (50 µM). Osteoclast progenitors were identified by staining for TRAP activity. (D) Quantitative analysis of osteoclast progenitors following in vitro culture. Experiment conducted on three independent occasions.

 
To validate our biochemical results, PD98059, a pharmacological inhibitor of Mek within the Ras-Erk signaling pathway, was utilized in the osteoclast culture and the effects on f-actin organization were examined. Similar to our previous data, we found that the Nf1+/– cells produced increased numbers and larger osteoclasts (Fig. 6C, a and b). Upon culture with PD98059 (50 µM), the size and the number of osteoclasts observed in the Nf1+/– cultures were reduced and identical to that observed in the WT cultures (Fig. 6C, c and d, and shown quantitatively in Fig. 6D). These data suggest that inhibition of the Ras-Erk signaling cascade has the capacity to normalize the gain-of-function of the Nf1+/– osteoclasts. A parallel study was performed to evaluate the effect of the PI3K inhibitor, Ly294002, on osteoclast formation from WT and Nf1+/– mice. Although multiple concentrations of Ly294002 were examined, we were unable to identify a Ly294002 concentration that preferentially inhibited the increased Nf1+/– osteoclast development without inducing cell death in both the Nf1+/– and WT cultures (data not shown). However, previous genetic studies performed in our laboratory demonstrated that genetic deletion of the regulatory subunit of PI3K, p85{alpha}, is indeed able to correct the increased>Nf1+/– osteoclast development (34). These previous data, along with the correction of M-CSF-stimulated Erk and Akt activation upon genetic deletion of Rac1, suggest that hyperactivation of Rac1-GTPase activity in the Nf1+/– osteoclasts contributes to the hyperactivation of the Ras-PI3K-Erk signaling cascade, thus implying that the pharmacologic inhibition of Rac1 may provide a rational therapeutic target for osteoporosis, particularly in NF1 patients.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
NF1 is the most common genetic disease in man with a predisposition to cancer. One in 3500 individuals worldwide is born with NF1. Despite NF1 being considered a neurocristopathy, at least 50% of NF1 patients have both generalized and focal skeletal abnormalities. Three distinct skeletal abnormalities (kyphoscoliosis, pseudoarthrosis and osteoporosis) are particularly debilitating. Though the risk of osteoporosis in the general population is well documented, multiple recent reports have provided evidence that greater than 30% of NF1 patients develop osteoporosis. Additionally, although osteoporotic fractures are an important public health problem in the aging population, osteoporosis is an emerging phenotype observed even in young individuals and, consequently, NF1 patients are likely even at greater risk than the general population (18,20,21,28). Therapies for prevention of fragility fractures are limited in scope, efficacy and acceptability to patients. Treatment of osteoclasts with the nitrogen-containing bisphosphonates, the current treatment of choice for erosive bone diseases, causes profound cytoskeletal disruption, including flattening of the ruffled border, consistent with Rac-GTPase inhibition (65,66). One potential mechanism of bisphosphonates is inhibition of Rac-GTPase function due to lack of post-translational prenylation as a result of downregulated isoprenoid biosynthesis (66,67), leading us to hypothesize that the Rac-GTPases may contribute to the observed Nf1+/– osteoclast gain-of-function. However, although bisphosphonates are the standard of care in the treatment of osteoporosis, Paget's disease of bone, and bone metastases, clinical compliance is limited due to complex administration instructions, significant gastrointestinal side effects when taken orally, and the need for intravenous injection of certain formulations. Furthermore, the effect of long-term therapy in children is unknown. Efforts to identify new, more effective treatments for osteoporosis, and to refine/optimize existing therapies are an active focus of many laboratories.

We have recently provided evidence that haploinsufficiency of NF1 (Nf1) in both humans and mice, respectively, results in increased osteoclast differentiation and f-actin associated functions, including migration, adhesion and bone resorption (34). Additionally, previous studies in our laboratory utilizing HSC-derived mast cells demonstrated that genetic deletion of Rac2 is able to normalize the gain-of-function mast cell phenotype induced by Nf1 haploinsufficiency and hyperactivation of the Ras/PI3K/Rac pathway (55,60). As osteoclasts are also an HSC-derived myeloid cell type and as on potential mechanism of bisphosphonate therapy is inhibition of Rac-GTPase function, we hypothesized that Rac-GTPases contribute to the osteoclast aberrancies observed in Nf1+/– mice and humans with NF1. To evaluate whether haploinsufficiency of Nf1 leads to alteration of Rac GTPases, Rac1 and Rac2-GTPase activity was measured in WT and Nf1+/– osteoclast progenitors. Our data indicate that Nf1 haploinsufficient osteoclasts exhibit hyperactivation of Rac1-GTPase. Consistent with the biochemical findings, functional studies utilizing genetic deletion of Rac1 revealed a crucial role of Rac1 in promoting increased osteoclastogenesis in the pathological Nf1+/– phenotype. The critical role of Rac1 in osteoclast function is emphasized by the fact that in spite of constitutive activation of Rac2-GTPase in the Nf1+/–; Rac1–/– osteoclasts, the Nf1+/– gain-of-function phenotype is corrected by genetic deletion of Rac1. These findings are significant as they reveal that the Rac GTPases contribute non-redundant functions in various myeloid cell types and imply that blocking Rac1 function, while sparing that of Rac2, may provide a level of specificity to erosive bone disease therapies. These findings are consistent with the very recent report published by Wang et al. (58).

The Ras-PI3K-Erk signaling cascade has been shown to be critical in physiologic osteoclast function and bone resorption (35–39) and, furthermore, hyperactivation of these pathways has been observed in disease models of Paget's disease of bone, bone metastasis, wear particle-induced osteolysis following arthroplasty, and NF1 (34,44–47). Congenital disorders bearing phenotypic overlap with NF1, including Noonan syndrome, Noonan-like/multiple giant cell lesion syndrome, LEOPARD syndrome, Costello syndrome and Cardio-facio-cutaneous syndrome, carry germline mutations within key genes contributing to hyperactivation of the Ras-Raf-Mek-Erk signaling cascade, implying that hyperactivation of the Ras-Rho-GTPase-Erk signaling cascade may contribute to the skeletal disorders observed in these developmental disorders, similar to that of NF1. Importantly, we show that genetic disruption of Rac1 in Nf1+/– osteoclasts corrects hyperactivation of Erk and Akt. To confirm these biochemical results, we also demonstrate that pharmacological inhibition of Mek (PD98059) is able reduce osteoclast development and belt formation. CDC42, a related RhoGTPase, has also been previously found to regulate podosome-mediated neutrophil motility via MAPK pathways (63). While we were unable to identify a concentration of Ly294002 that was able to normalize the increased Nf1+/– osteoclast development while still permitting cell viability, we have previously shown that genetic deletion of p85{alpha}, a regulatory subunit of PI3K, is able to normalize the increased Nf1+/– osteoclast development. The class IA PI3Ks are a group of heterodimeric lipid kinases composed of a p85 regulatory subunit (p85{alpha}, p55{alpha}, p50{alpha}, p85β or p85{gamma}) and a p110 catalytic subunit (p110{alpha}, p110β or p110d) (68,69). The non-specific effect of Ly294002 on Nf1+/– osteoclast development suggests that the PI3K regulatory subunits may play non-redundant functions in osteoclast development. The genetic, biochemical and pharmacologic data, taken together, support the hypothesis that Rac1-GTPase hyperactivation in Nf1+/– osteoclasts contributes to the hyperactivation of the Ras-PI3K-Erk signaling pathway. Collectively, these studies not only define a novel molecular mechanism that underlies the pathological osseus anomalies observed in NF1, but also imply that novel therapeutics designed to inhibit osteoclast-specific Rac1-GTPase activity may provide a rational strategy for the treatment of dysplasic and erosive bone diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
FUNDING
 REFERENCES
 
Animals and materials
Nf1+/– mice were obtained from Tyler Jacks at the Massachusetts Institute of Technology (Cambridge, MA, USA) in a C57BL/6J.129 background and backcrossed for 13 generations into a C57BL/6J strain (70). Rac2–/– mice were backcrossed for 12 generations into a C57BL/6J strain as previously described (71). Rac1 conditional (Rac1flox/flox) and null alleles were generated by Dr Kwiatkowski as previously reported (72,73). The conditional Rac1 allele contains loxP sites flanking exon 1; upon cre-mediated excision this allele generates a null allele. Rac1 conditional knockout mice were crossed with MxCre-transgenic C57BL/6 mice. Animals received three intraperitoneal injections at 3 months of age with 300 µg poly I:-poly C (poly I:C) (Sigma, St Louis, MO) diluted in sterile phosphate-buffered saline (PBS). Mice were sacrificed for bone marrow cell harvest 48 h after the last injection. These studies were conducted with a protocol approved by the Indiana University Laboratory Animal Research Center. The Nf1, Rac1flox/floxMxcre+ and Rac2 alleles were genotyped by PCR, as previously described (62,73,74). Chemicals were purchased from Sigma (St Louis, MO) unless otherwise stated.

Clonogenic progenitor assays
Murine bone marrow mononuclear cells (BMMNCs) were cultured in 0.3% agar containing DMEM, L-asparagine, DEAE dextran and 20% fetal calf serum in 1 ml aliquots in 35 mM petri dishes, as previously described (34). To identify osteoclasts, colonies were fixed with a citrate/acetone/formaldehyde solution and histochemically stained for TRAP, as described (34).

Generation of murine osteoclasts
BMMNCs from all the experimental groups of mice were cultured in {alpha}–MEM supplemented with 10% fetal bovine serum (FBS, Biomeda, Chess Drive Foster City, CA) in the presence of human recombinant RANKL (50 ng/ml, Peprotech, Rocky Hill, NC) and murine recombinant M-CSF (30 ng/ml, Peprotech) for 7 days as previously described (34). The medium was changed every 3 days. Osteoclasts were placed on plastic dishes, washed with {alpha}-MEM and treated with 0.1% collagenase and 0.2% dispase to remove stromal cells. To identify osteoclasts at the end of culture, adherent cells were fixed with 10% formaldehyde in PBS, treated with ethanol–acetone (50:50) and stained for TRAP, as described (34), and imaged using a Nikon TE2000-S microscope (Nikon Inc.). Images were taken by a QImaging camera and QCapture-Pro software (Fryer Company Inc., Cincinnati, OH). Multinucleated, TRAP-positive cells containing more than three nuclei were scored as mature osteoclasts.

Thymidine incorporation assay
Proliferation was assessed by conducting a thymidine incorporation assay on osteoclast progenitors of the indicated genotypes. Briefly, cells were washed and starved in 0.2% BSA with or without any growth factors for 6–7 h. 5x104 cells were plated in a 96-well plate in 20`0 µl of complete medium containing 10% FBS and 30 ng/ml of M-CSF in {alpha}-MEM. Cells were cultured for 48 h and subsequently pulsed with 1.0 µCi of [3H] thymidine for 6–8 h. Cells were harvested using an automated 96-well cell harvester (Brandel, Gaithersburg, MD) and thymidine incorporation was determined as counts per minute.

Osteoclast migration assay
Migration of osteoclasts was evaluated using a transwell assay as described with minor modifications (34,62,75). To assure the number of loaded cells into each transwell, BMMNCs previously cultured in M-CSF and RANKL for 5 days were lifted from the plates by adding 0.05% trypsin and 0.2% EDTA.4Na in HBSS and scored to identify TRAP+ cells. Equivalent numbers of cells were loaded onto the upper chamber of an 8µm polycarbonate transwell coated with vitronectin for 15 h in a humidified incubator at 37°C, and the lower chamber was added with {alpha}-MEM, 0.1% bovine serum albumin (BSA) and M-CSF (30 ng/ml). After 4 h incubation, cells that were migrated to the bottom of chambers were stained for TRAP and TRAP+ cells per field were then counted (Empire Imaging Systems, Plattsburgh, NY).

Bone resorption assay
Following migration to the local area, osteoclasts form a specialized cell–extracellular matrix to initiate degradation of bone matrix by secreting proteinases (61). This bone resorptive function is assessed in vitro by culturing osteoclasts on dentine slices and examining the number and area of ‘pits’ that are resorbed by osteoclasts as described previously (34). Single cell suspensions of purified osteoclasts were seeded onto dentine slices (34,76) (ALPCO Diagnostic, Windham, NH), incubated at 37°C, 5% CO2 in the presence of M-CSF and RANKL. Following 7 days culture, the slices were rinsed with PBS, then left overnight in 1 M ammonium hydroxide and stained with 1% toluidine blue in 0.5% sodium tetraborate solution. The number of resorptive areas or ‘pits’ per LPF on each bone slice was counted using reflective light microscopy. The area (mm2) of each pit was evaluated by measuring widthxlength using QCapture Pro (Version 5.1) by an investigator who was blinded to the experimental groups.

Evaluation of biochemical activities
Erk phosphorylation were determined by western blot using phospho-specific antibody for Erk (New England Biolabs, Beverly, MA), as described previously (34,77). Osteoclasts were deprived of serum and growth factors for 47emsp14;h followed by stimulation with 30 ng/ml M-CSF for various amounts of time. Densitometry of individual bands was conducted using NIH Image software. To examine the Rac1 GTPase and Rac2 GTPase activity, Rac activation was determined using a PAK pulldown assay (Upstate Biotechnology, cat. #17-283 for Rac1 activation and cat. # 17-369 for Rac2 activation), as previously described (55,77,78).

Immunofluorescence microscopy
To evaluate the cytoskeletal organization in Nf1+/– osteoclasts, immunofluorescence microscopy was used to examine the podosome formation as previously described (48,50). Briefly, osteoclasts were grown on coverslips, washed with PBS and permeabilized with 0.01% saponin in 80 mM Pipes, pH 6.8, 5 mM EGTA, 1 mM MgCl2 for 5 min at room temperature. The cells were fixed in 3% paraformaldehyde in PBS for 20 minutes and quenched with 50 mM NH4Cl for 10 min. The cells were then washed in 2% BSA with 0.01% saponin in PBS for 5 min to block nonspecific binding. FITC-conjugated phalloidin (Sigma, St Louis, MO) was used to incubate with the permeabilized cells for 1 h at room temperature. After washing three times with the same buffer, nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Slides were washed and then mounted with 80% glycerol in PBS. Confocal laser scanning microscopy was performed to obtain and analyze the immunofluorescence images with a Leica TCS-SP unit equipped with argon-krypton lasers (Leica Microsystems Heidelberg GmbH) and a 40x oil immersion lens.

Statistical analysis
Two-tailed Student's t-test and ANOVA were used to evaluate statistical difference. P-values less than 0.05 were considered significant.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
REFERENCES
 
This work was supported by NF043032 (F.-C.Y.).


   ACKNOWLEDGEMENTS
 
We thank D. Wade Clapp for critical reading of the manuscript. We also thank Marilyn Wales for administrative support.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} These authors contributed equally to this to this work. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 

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