Repositioning Potential of PAK4 to Osteoclastic Bone Resorption

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1 ORIGINAL ARTICLE JBMR Repositioning Potential of PAK4 to Osteoclastic Bone Resorption Sik-Won Choi, 1 Jeong-Tae Yeon, 2 Byung Jun Ryu, 1 Kwang-Jin Kim, 2 Seong-Hee Moon, 1 Hyuk Lee, 3 Myeung Su Lee, 4 Sam Youn Lee, 5 Jin-Chul Heo, 6 Sang-Joon Park, 7 and Seong Hwan Kim 1 1 Laboratory of Translational Therapeutics, Pharmacology Research Center, Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon, South Korea 2 Department of Pharmacy, Sunchon National University, Suncheon, South Korea 3 Medicinal Chemistry Research Center, Korea Research Institute of Chemical Technology, Daejeon, South Korea 4 Division of Rheumatology, Department of Internal Medicine, Wonkwang University, Iksan, Jeonbuk, South Korea 5 Department of Cardiac and Thoracic Surgery, Wonkwang University, Iksan, Jeonbuk, South Korea 6 Pharmacology Research Center, Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon, South Korea 7 Department of Histology, College of Veterinary Medicine, Kyungpook National University, Daegu, South Korea ABSTRACT Drug repositioning is a rational approach for expanding the use of existing drugs or candidate drugs to treat additional disorders. Here we investigated the possibility of using the anticancer p21-activated kinase 4 (PAK4)-targeted inhibitor PF to treat osteoclast-mediated disorders. PAK4 was highly expressed in bone marrow cells and was phosphorylated during their differentiation into osteoclasts, and osteoclast differentiation was significantly inhibited by the dominant negative form of PAK4 and by PF Specifically, PF significantly inhibited the fusion of preosteoclasts, the podosome formation, and the migration of preosteoclasts. PF also had in vivo antiresorptive activity in a lipopolysaccharide-induced bone erosion model and in vitro antiosteoclastogenic activity in the differentiation of human bone marrow derived cells and peripheral blood mononuclear cells into osteoclasts. These data demonstrate the relevance of PAK4 in osteoclast differentiation and the potential of PAK4 inhibitors for treating osteoclast-related disorders American Society for Bone and Mineral Research KEY WORDS: OSTEOCLAST; PAK4 INHIBITOR; DRUG REPOSITION; PODOSOME; OSTEOCLAST FUSION Introduction Drug repositioning refers to the strategy of expanding the use of approved drugs or drug candidates that are in the development stage to treat new indications. (1 3) Drug repositioning is also referred to as drug redirecting, reprofiling, retasking, and indication expansion. From the viewpoint of a pharmaceutical company, a drug repositioning strategy is costeffective because it can reduce the time and expense involved in developing new drug candidates. For basic researchers, when the concept of drug repositioning is applied in the early stage of drug discovery, target-specific drugs (or candidate drugs) can be used as tools to validate the relevance of the target molecule in other disorders, to identify new first-in-class mechanisms to better understand disorders, and to evaluate the usefulness of pharmacological inhibition of the target of interest. Importantly, drug repositioning for novel indications or new diseases necessitates in-depth study of biological mechanisms and a clear definition of the new indication. Consequently, these efforts create new pipelines by entering clinical trials. Therefore, drug repositioning is a rational approach that not only clarifies whether a drug target is involved in an additional indication but also involves the development of an active drug for a purpose other than the original indication. Bone is a basic and dynamic unit of the skeletal system that is needed for structural support, to protect organs, for mineral homeostasis, and for locomotion. Bone remodeling is achieved by the repeated cyclic process of osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Accordingly, an imbalance of bone remodeling due to increased numbers and/or overactivation of osteoclasts leads to osteoclast-related diseases such as osteoporosis, rheumatoid arthritis, Paget s disease, periodontal disease, and cancer bone metastasis. (4 7) Because patients with osteoclast-related diseases are at higher risk of fractures, the economic burdens for hospitalizations due to fractures have become a serious public health Received in original form September 12, 2014; revised form January 19, 2015; accepted January 23, Accepted manuscript online January 31, Address correspondence to: Seong Hwan Kim, PhD, Laboratory of Translational Therapeutics, Pharmacology Research Center, Drug Discovery Division, Korea Research Institute of Chemical Technology, P.O. Box 107, Yuseong-gu, Daejeon , South Korea. hwan@krict.re.kr Additional Supporting Information may be found in the online version of this article. Journal of Bone and Mineral Research, Vol. 30, No. 8, August 2015, pp DOI: /jbmr American Society for Bone and Mineral Research 1494

2 issue. (8) Therefore, pharmaceutical inhibition of osteoclast differentiation and/or activity can be used to treat patients with osteoclast-related diseases to mitigate the extent of bone loss and to reduce bone loss related fractures. The differentiation of hematopoietic stem cells (monocytes and macrophages) into multinucleated osteoclasts is a complex multistep process that involves cell differentiation, migration, fusion, and resorption. In bone marrow, osteoclast differentiation is directly induced by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and receptor (9) activator of nuclear factor-kb ligand (RANKL). M-CSF and RANKL trigger the differentiation of osteoclast precursors into mononuclear osteoclasts (preosteoclasts) and induce them to migrate until they attach to the bone surface. Mononuclear osteoclasts then fuse with each other to form giant multinucleated osteoclasts that subsequently mediate bone resorption. (10,11) In response to RANKL, c-fos, a protein that is part of the AP-1 transcription factor complex, is induced in the early-to-middle stages of osteoclast differentiation, and ``nuclear factor of activated T cells, cytoplasmic 1'' (NFATc1) is induced in the middle-to-late stages of osteoclast differentiation. The temporal induction of these transcription factors plays a role in the transcriptional regulation of specific genes related to osteoclast differentiation, migration, fusion, and/or resorption. p21-activated kinases (PAKs) are part of the serine/threonine kinase family and comprise six proteins that can be subdivided into two groups based on sequence similarity and functional characteristics: PAK1 to PAK3 (group I) and PAK4 to PAK6 (12) (group II). PAKs play important roles in cell morphology, cytoskeletal reorganization, apoptosis, survival, angiogenesis, and metastasis. (13) Among them, PAK4 is overexpressed in various types of human cancers, including colorectal, breast, ovarian, and lung cancers. (14) Nothing is known about the function of PAKs in osteoclasts, but a recent study shed light on their potential roles in the control of actin cytoskeletal reorganization and podosome formation in macrophages. Specifically, PAK4 localizes to podosome cores in macrophages, and its kinase activity regulates the size and number of individual podosomes. (15) In addition, PAK4 overexpression induces actin polymerization, the formation of filopodia, and cell migration; notably, PAK4-dependent changes in the actin cytoskeleton require its kinase activity. (16,17) Interestingly, actin cytoskeletal reorganization and podosome formation are closely associated with cell fusion and migration during osteoclast differentiation, suggesting the possible involvement of PAK4 in this process. Taken together, these observations prompted us to evaluate the relevance of PAK4 to osteoclast differentiation and the antiosteoclastogenic action of its pharmacological inhibition by PF (15,17,18) PF was developed by Pfizer (New York, NY) as an anticancer agent, (19 21) but its clinical phase I trial of escalating oral doses in patients with advanced solid tumor (ClinicalTrials. gov Identifier: NCT ) was terminated prematurely in 2011 because of its undesirable pharmacokinetic characteristics and the lack of an observed dose-response relationship. However, because there were no safety concerns that contributed to the study termination, PAK4 inhibitors, including derivatives of PF , are still being developed by Pfizer and other companies for treating cancers and Alzheimer s disease-related dementia. Here, we demonstrated the crucial role of PAK4 in osteoclast differentiation using its inhibitor PF Materials and Methods Chemicals and reagents PF {N-[(1S)-2-(dimethylamino)-1-phenylethyl]-6,6-dimethyl-3-[(2-methylthieno[3,2-d]pyrimidin-4-yl) amino]-4,6- dihydropyrrolo[3,4-c]pyrazole-5(1h)-carboxamide); Fig. 1A} was synthesized as a yellowish solid as described previously (WO ) and was characterized as described in Supporting Fig. 1. Its chemical formula is C 25 H 30 N 8 OS, and its molecular weight is Its purity was over 98% as determined by liquid chromatography/mass spectroscopy (LC/MS), with one positive ion of Da (predicted, Da, MþH, protonated molecular ion). Mouse soluble M-CSF was purchased from PeproTech. Human M-CSF and mouse/human RANKL were purchased from R&D Systems. Antibodies were purchased from the following companies: antibodies against NFATc1, c-fos, actin, vinculin, cortactin, and cofilin, and secondary antibodies, from Santa Cruz Biotechnology; antibodies against PAK4 and p-cofilin from Cell Signaling; antibodies against p-pak4 and p-cortactin from Bioss; antibody against the myc-epitope from Abcam; and AlexFluor 488-labeled goat anti-mouse IgG and AlexFlour 633-labeled goat anti-rabbit IgG from Invitrogen. Preparation of preosteoclasts All experiments were carried out as described with the indicated modifications. (22) All animal procedures were performed according to the guidelines of the Institutional Animal Care and Use Committee of the Korea Research Institute of Chemical Technology (Protocol ID No. 7D-M1). Five-week-old male ICR mice (Damul Science Co.) were maintained in a room that was illuminated daily from 7:00 am to 7:00 pm (a 12-hour:12-hour light/dark cycle) under controlled temperature (23 C 1 C) and ventilation (10 to 12 times per hour). Humidity was maintained at 55% 5%, and the mice had free access to a standard animal diet and tap water. Mouse bone marrow cells (BMCs) were obtained from 5-week-old male ICR mice by flushing the femurs and tibias with a-mem containing antibiotics (100 units/ml penicillin, 100 mg/ml streptomycin). BMCs were cultured in a-mem containing 10% FBS and M-CSF (10 ng/ml) for 1 day on culture dishes. Nonadherent BMCs were plated on Petri dishes and cultured for 3 days in the presence of M-CSF (30 ng/ml). After nonadherent cells were washed away, adherent cells were used as bone marrow-derived macrophages (BMMs). According to the Ethics Guidelines for Wonkwang University Hospital and after approval by the Institutional Review Board (IRB; HRBR- 014), human BMCs were isolated and cultured for 3 days with human M-CSF (50 ng/ml). CD14-positive human peripheral blood mononuclear cells (hpbmcs) were purchased from Zen-Bio Inc. Osteoclast differentiation BMMs were maintained in a-mem supplemented with 10% FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin in a humidified atmosphere of 5% CO 2 at 37 C. The medium was changed every 3 days. To differentiate BMMs into osteoclasts, BMMs were cultured for 4 days with M-CSF (30 ng/ml) and RANKL (10 ng/ml). Multinucleated osteoclasts were observed after 3 to 4 days. hpbmcs were cultured for 7 days with human M-CSF (50 ng/ml), and adherent hpbmcs were further cultured with M-CSF (50 ng/ml) and RANKL (25 ng/ml) for 14 days. Human primary bone marrow cells (hbmcs) were cultured for 3 days with human M-CSF (50 ng/ml) and then incubated for 7 days with RANKL (50 ng/ml) and M-CSF (50 ng/ml). Journal of Bone and Mineral Research REPOSITIONING POTENTIAL OF PAK4 TO OSTEOCLASTIC BONE RESORPTION 1495

3 Fig. 1. The effect of PAK4 activity and PF on osteoclast differentiation. (A) The structure of PF (B) GeneChip-based mrna expression analysis of PAKs in BMMs treated with RANKL for the indicated time. (C) Expression levels of PAK4 and phosphorylated PAK4 proteins during RANKLinduced osteoclast differentiation. (D) The expression of NFATc1 and vinculin in BMMs transduced with the retroviral WT, S476E, or K352M forms of PAK4. pmx-ires-puro-myc was used as a control. (E) Transduced BMMs were cultured for 4 days with M-CSF (30 ng/ml) and RANKL. After TRAP staining, the number of TRAP þ -MNCs was counted (n ¼ 3). (F) The effect of PF on the RANKL-induced formation of TRAP þ -MNCs was visualized and evaluated by counting the number of TRAP þ -MNCs (G)(>3 nuclei, left panel; >10 nuclei, right panel, n ¼ 3). (H) TRAP activity was also measured. ###p < (versus the negative control); **p < 0.01 and ***p < (versus the RANKL-treated group). (I) The effect of PF on BMM viability (n ¼ 3). Data are expressed as mean SD and are representative of at least three experiments. Western blot analysis Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Elpis Biotech). Proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Blots were probed with antibodies, developed using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce), and visualized with the LAS-3000 luminescent image analyzer (Fuji Photo Film Co., Ltd.). Real-time PCR and microarray analysis Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer s recommended protocol CHOI ET AL. Journal of Bone and Mineral Research

4 First-strand cdna was synthesized using the Omniscript RT kit (Qiagen) with 1 mg of total RNA. SYBR green-based qpcr was performed with the Stratagene Mx3000 Real-Time PCR system and Brilliant SYBR Green Master Mix (Stratagene). Supporting Table 1 lists the primers used in this study. Microarray analysis was performed by ebiogen (Seoul, Korea). Retrovirus-based functional study The PAK4 gene was cloned by RT-PCR using total RNA isolated from BMMs. The site-directed mutagenesis of PAK4 was performed based on overlap extension PCR. PAK4 (WT), PAK4 (S476E), and PAK4 (K352M) were cloned into the retroviral vector pmx-puro-c-myc (Cell Biolabs, Inc.). The cloned sequences are shown in Supporting Fig. 2. Retroviral packaging was performed by transfecting the plasmids into Plat-E cells (platinum-e retrovirus packaging cell line; Cell Biolabs, Inc.) using Lipofectamine 2000 (Invitrogen). Viral supernatants were collected 2 days after transfection and added to BMMs in the presence of polybrene (10 mg/ml). After infection, BMMs were cultured overnight, detached using StemPro Accutase Cell Dissociation Reagent (Invitrogen), and then cultured with M-CSF (30 ng/ml) and puromycin (2 mg/ml) for 2 days. Puromycin-resistant BMMs were differentiated into osteoclasts by culturing them with M- CSF (30 ng/ml) and RANKL (10 ng/ml) for 4 to 5 days. For overexpression of constitutively active (CA)-NFATc1, pmx containing CA-NFATc1 was transiently transfected into Plat-E cells with Lipofectamine 2000 according to the manufacturer s protocol. Viral supernatants were collected 2 days after transfection and added to BMMs in the presence of polybrene (10 mg/ml) for 8 hours. After infection, BMMs differentiated into osteoclasts during 3 days of culture with M-CSF (30 ng/ml) and RANKL (10 ng/ml). Tartrate-resistant acid phosphatase staining and activity assay Mature osteoclasts were visualized by staining for tartrateresistant acid phosphatase (TRAP), a biomarker of osteoclast differentiation. Briefly, multinucleated osteoclasts were fixed with 3.7% formalin for 10 min, permeabilized with 0.1% Triton X- 100 for 10 min, and stained with TRAP solution (Sigma-Aldrich). TRAP-positive multinucleated osteoclast cells (TRAP þ -MNCs) (3 nuclei) were counted. To measure TRAP activity, MNCs were fixed in 3.7% formalin for 5 min, permeabilized with 0.1% Triton X-100 for 10 min, and treated with TRAP buffer (100 mm sodium citrate ph 5.0, 50 mm sodium tartrate) containing 3 mm p- nitrophenyl phosphate (Sigma-Aldrich) at 37 C for 5 min. Reaction mixtures in the wells were transferred to new plates containing an equal volume of 0.1 N NaOH and the optical density was determined at 405 nm. Cell proliferation assay BMMs were plated in a 96-well plate at a density of cells/ well in triplicate. After treatment with M-CSF (30 ng/ml) and PF , the cells were incubated for 3 days and cell viability was measured using the Cell Counting Kit 8 (CCK-8) according to the manufacturer s protocol. Bone pit formation analysis BMMs that were differentiated into preosteoclasts on BioCoat Osteologic MultiTest slides in the presence of M-CSF (30 ng/ml) and RANKL (10 ng/ml) were treated with PF for 24 hours. TRAP-stained cells on the slides were photographed under a light microscope at 40 magnification. To measure the area of the resorbed pits, the slides were washed with PBS, treated with 5% sodium hypochlorite for 5 min, washed with PBS buffer, dried, and photographed under a light microscope. Quantification of resorbed areas was performed using the ImageJ program (NIH; Immunofluorescence staining Cells cultured on black clear glass plates were fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100 for 10 min, and incubated with primary antibodies for 2 hours. Cells were washed with PBS containing 0.1% bovine serum albumin (BSA), incubated with Alexa Fluor (488 or 633)-conjugated secondary antibodies for 1 hour, and then incubated with 4,6-diamidino-2- phenylindole (DAPI). To detect F-actin, cells were stained with phalloidin-fitc (Sigma-Aldrich). Stained cells were photographed under a fluorescence microscope (Olympus IX51) and analyzed using Cellomics Arrayscan (Thermo Scientific). Invasion and migration assays The ability of preosteoclasts to pass through a gelatin-coated membrane was measured in a Boyden chamber using an 8-mmpore polycarbonate membrane (Neuro Probe) coated with gelatin (Sigma-Aldrich) that was diluted to 0.1 g/l in 1% acetic acid. Preosteoclasts cultured on a Petri dish for 2 days with M- CSF and RANKL were detached using trypsin and resuspended in the culture medium. Culture media (30 ml) containing various concentrations of PF were applied to the lower chamber, and resuspended cells (50 ml, cells/well) were seeded in the upper chamber. After incubation for 9 hours at 37 C, cells on the upper surface of the membrane were carefully removed with a cotton swab, and cells that had migrated through the gelatin to the lower surface of the membrane were fixed and stained with Diff-Quick (Dade Behring). The number of invaded cells was counted in random areas of the membrane. To measure the migration of preosteoclasts, a wound-healing assay was performed. BMMs were plated on 96-well plates (Essen ImageLock; Essen Instruments) and incubated with M-CSF and RANKL for 3 days to induce differentiation into preosteoclasts. A wound was made with a wound scratcher (Essen Instruments) and PF was added. Cell confluence in the wound area was monitored and analyzed with the Incucyte Live-Cell Imaging System and software (Essen Instruments) every 3 hours for 30 hours by comparing the mean relative wound density of six replicates in each experiment. In vivo lipopolysaccharide-induced bone erosion In vivo experiments using the lipopolysaccharide (LPS)-induced osteoporotic animal model were carried out according to the Ethics Guidelines of the Korea Research Institute of Chemical Technology (Protocol ID No. 7D-M2). The protocol was approved by the Institutional Committee (Approval No D-04-03). The 5-week-old ICR mice were divided into four groups of 5 mice. Mice were injected intraperitoneally with PF (0.5 or 1 mg/kg body weight) or PBS (vehicle control) 1 day before injection of LPS (10 mg/kg body weight). PF or PBS was injected intraperitoneally every day for 8 days, and LPS was injected intraperitoneally on days 1 and 4. All mice were euthanized 8 days after the initial LPS injection, and the left femurs of all animals were scanned using high-resolution Journal of Bone and Mineral Research REPOSITIONING POTENTIAL OF PAK4 TO OSTEOCLASTIC BONE RESORPTION 1497

5 micro computed tomography (mct) (SKYSCAN 1173). The images were analyzed using CTvol (3D visualization) and DataViewer (SKYSCAN). Bone histomorphometric analyses were performed with the mct data using the CTAn software provided by the SKYSCAN analysis tool. Bone mineral density (BMD), bone volume/tissue volume (BV/TV or bone volume fraction), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were measured to assess the trabecular bone microstructure of the femur. For histologic analysis, femurs were isolated, fixed with 4% paraformaldehyde (Sigma-Aldrich) for 1 day and decalcified with 12% EDTA. Decalcified bones were embedded in paraffin, cut to a thickness of 6 mm and stained with hematoxylin and eosin (H&E) to examine bone phenotype. Other sections were stained with TRAP to visualize osteoclasts. The number of osteoclasts was counted in TRAP-stained sections. A parameter of osteoclast numbers, osteoclast surface per bone surface (Oc.S/BS), was quantified by using the ImageJ. Stained bones were photographed under a light microscope. All bone histomorphometry were described in accordance with standard criteria. (23,24) Statistical analysis All quantitative values are presented as mean SD. Each experiment was performed three to five times, and the results from one representative experiment are shown. Statistical differences were analyzed using the Student s t test. A value of p < 0.05 was considered significant. Results PAK4 is highly expressed in bone marrow cells and is phosphorylated by RANKL during their differentiation into osteoclasts Microarray analysis revealed that, of the PAKs, PAK4 was highly expressed in bone marrow cells, and its expression was maintained during RANKL-induced osteoclast differentiation (Fig. 1B). The expression level of the PAK4 protein was also maintained, but its phosphorylation increased over time during osteoclast differentiation (Fig. 1C). Osteoclast differentiation was confirmed by evaluating the expression levels of the c-fos and NFATc1 proteins over time (Fig. 1C). PAK4 kinase activity is essential for RANKL-induced osteoclast differentiation In order to verify the relevance of PAK4 to osteoclast differentiation, a retrovirus-based functional study was performed. Specifically, the myc-tagged native form (WT) of PAK4, the constitutive active form (S476E) of PAK4, or the dominant negative form (K352M) of PAK4 was overexpressed in BMMs (Fig. 1D). RANKL-induced formation of TRAP þ -MNCs was significantly increased by the constitutively active form of PAK4 (S476E) compared to the native PAK4 (WT) and was decreased by the dominant negative form (K352M) (Fig. 1E). Pharmacological inhibition of PAK4 by PF inhibits RANKL-induced osteoclast differentiation The possibility that PAK4 kinase activity might regulate osteoclast differentiation further prompted us to evaluate the antiosteoclastogenic activity of the PAK4 inhibitor PF Furthermore, PF and its derivatives are still being developed (Supporting Table 2), but the effect of PF in osteoclastogenesis has not been reported. PF strongly inhibited the RANKL-induced formation of TRAP þ -MNCs in a dose-dependent manner at nanomolar concentrations, as shown in Fig. 1F and G. Moreover, there were fewer TRAP þ - MNCs with >10 nuclei when cells were treated with PF PF also significantly attenuated the activity of TRAP, a biomarker of osteoclast differentiation, in a dose-dependent manner (Fig. 1H). However, PF did not affect the survival of BMMs, indicating that the antiosteoclastogenic activity of PF was not due to cytotoxicity (Fig. 1I). Furthermore, at the concentrations used in this study, PF did not affect the osteoblast differentiation and mineralization of mouse neonatal calvarial preosteoblasts (Supporting Fig. 3). These results suggest that pharmacological inhibition of PAK4 by the specific inhibitor PF at nanomolar concentrations strongly inhibited RANKL-mediated osteoclast formation without any changes in osteoblast differentiation. PF inhibits cell fusion in the late stage of osteoclast differentiation To better understand how PF inhibits osteoclast differentiation, we investigated its effects on RANKL-triggered early signaling pathways. PF did not affect the activation of early signaling molecules, such as Akt and MAP kinases (Supporting Fig. 4). Moreover, RANKL activation of PAK4 occurred in the late stage of osteoclast differentiation (Fig. 1C). Therefore, we next asked whether PF preferentially affected the late stages of osteoclast differentiation. To address this question, we reevaluated the antiosteoclastogenic activity of PF by treating cells at four time points as shown in Fig. 2A. There were significantly fewer TRAP þ -MNCs with >10 nuclei formed when cells were treated with PF for 24 hours in the early to middle stages of differentiation (1 to 2 days or 2 to 3 days after RANKL treatment), and their formation was almost completely inhibited when preosteoclasts were incubated for 24 hours with PF in the late stage of osteoclast differentiation (3 to 4 days after RANKL treatment; Fig. 2B). The inhibitory effect of PF on the formation of functionally activated TRAP þ -MNCs was confirmed by staining the actin rings and evaluating pit formation. Actin ring formation is the defining characteristic of MNCs, and bone pit formation is the defining characteristic of functionally activated osteoclasts. Incubation of preosteoclasts with 5 nm PF for 1 day inhibited the formation of giant osteoclasts with actin rings, as shown by staining with fluorescence-conjugated phalloidin (Fig. 2C). Also, because of the antiosteoclastogenic activity of PF , the areas of resorption were smaller when preosteoclasts were incubated with PF for 1 day (Fig. 2D, E). In the absence of PF , the synthetic carbonate apatite was fully resorbed by functionally activated osteoclasts. Because the late stage of osteoclast differentiation is important in the processes of cell fusion and osteoclast maturation, the antiosteoclastogenic activity of PF was confirmed by evaluating the mrna expression levels of osteoclast fusion/maturation-related proteins, including NFATc1 (Fig. 2F). In preosteoclasts, RANKL treatment strongly induced the expression of mrna coding for osteoclast fusion/maturation-related proteins; PF significantly attenuated mrna induction, with the exception of c-src mrna. Western blot analysis showed that PF strongly inhibited the expression of the NFATc1 protein in preosteoclasts 1498 CHOI ET AL. Journal of Bone and Mineral Research

6 Fig. 2. PF impairs the fusion of preosteoclasts. (A) According to the exposure schedule, BMMs were treated with vehicle (0.1% DMSO) or PF (5 nm) in the presence of M-CSF and RANKL. (B) After BMM differentiated into osteoclasts as described in A, TRAP þ -MNCs were photographed under a light microscope and the number of TRAP þ -MNCs (>10 nuclei) were counted (n ¼ 3). Each exposure period of PF was indicated as ``vehicle'' for the vehicle, ``0 1'' for 0-1 day, ``1-2'' for 1 2 day, ``2-3'' for 2 3 day, and ``3-4'' for 3 4 day. *p < 0.05, **p < 0.01, and ***p < versus the RANKL-treated group. (C) Preosteoclasts derived from BMMs by culture with M-CSF and RANKL for 3 days were treated with PF (5 nm) for 1 day with or without RANKL (10 ng/ml). The actin rings of mature osteoclasts were stained with phalloidin. (D) Preosteoclasts were cultured on carbonate apatite plates (BioCoat Osteologic MultiTest slides) for 1 day with or without PF (5 nm), fixed, permeabilized, stained with TRAP, and photographed under a light microscope (upper panel). After detaching the cells, slides were photographed under a light microscope (bottom) to observing the resorbed areas. (E) TRAP þ -MNCs (>10 nuclei) on the slides were counted, and the resorbed areas were quantified using the ImageJ program (n ¼ 3). ***p < versus the vehicle control. (F, G) The indicated mrna and protein expression levels were evaluated during preosteoclast differentiation into mature osteoclasts in the absence or presence of PF (5 nm) by real-time PCR (mrna) or Western blot analysis (proteins). The relative fold change of mrna expression level compared to the control was presented. *p < 0.05, **p < 0.01, and ***p < versus the vehicle control, n ¼ 3. Data are representative of at least three experiments. Journal of Bone and Mineral Research REPOSITIONING POTENTIAL OF PAK4 TO OSTEOCLASTIC BONE RESORPTION 1499

7 (Fig. 2G). PF mediated cellular inhibition of PAK4 in the preosteoclasts was also confirmed; without any change in the level of the PAK4 protein, PF strongly attenuated the RANKL-induced phosphorylation of PAK4 but not c-src (Fig. 2G). In addition, PF did not affect the RANKL-induced formation of TRAP þ -mononuclear preosteoclasts (Supporting Fig. 5A, B) or the expression levels of osteoclast differentiationrelated proteins, including c-fos, NFATc1, and TRAP, during the differentiation of BMMs into preosteoclasts (Supporting Fig. 5C, D). PF inhibits expression and activity of podosome-related proteins in preosteoclasts We next examined whether PF inhibited the expression and/or activity of podosome-related proteins, including vinculin, cortactin, and cofilin. As shown in Fig. 3A, RANKL induced the expression of vinculin, cortactin, and the active (phosphorylated) form of cortactin during osteoclast differentiation. Also, cofilin activation (dephosphorylation) was observed in the late stage of osteoclast differentiation. However, when PAK4 was inhibited (dephosphorylated) by PF , which was accompanied by the downregulation of NFATc1 during the transition from preosteoclasts to osteoclasts, the expression of vinculin and the activities of both cortactin and cofilin were also inhibited (Fig. 3B). The effects of PF on podosomes and on the F-actin-associated protrusions that link two mononuclear preosteoclasts for fusion were investigated by vinculin and F-actin staining, respectively. In addition to the activation of PAK4, the formation of protrusions in preosteoclasts was inhibited by PF (Fig. 3C, Supporting Fig. 6). Overexpression of NFATc1 counteracts the PF mediated inhibition of osteoclast differentiation The antiosteoclastogenic activity of PF might result from its potential to inhibit NFATc1 expression, which would subsequently affect the expression of podosome-related proteins. This hypothesis was evaluated by investigating whether the antiosteoclastogenic activity of PF could be countered by overexpressing NFATc1. Consistent with the result shown in Fig. 1F, fewer TRAP þ -MNCs were formed in the presence of 5 nm PF (upper images in Fig. 4A), but overexpression of NFATc1 dramatically overcame the antiosteoclastogenic action of PF (bottom images in Fig. 4A). The ability of NFATc1 overexpression to reverse PF induced inhibition of osteoclast differentiation was confirmed by counting the number of TRAP þ -MNCs and measuring TRAP activity (Fig. 4B, C). Interestingly, NFATc1 overexpression induced the phosphorylation of PAK4 and expression of the cortactin and vinculin proteins (Fig. 4D), effects that are similar to those of RANKL (Figs. 1C, 3A). However, only the level of the NFATc1-induced vinculin protein was maintained in NFATc1- induced MNCs even after PF treatment (Fig. 4D). These results suggest that PF inhibits RANKL-induced osteoclast differentiation by suppressing the expression/activity of NFATc1 and its target molecule, vinculin. The downregulation of NFATc1 and vinculin by the dominant negative form (K352M) was consistent with this notion (Fig. 1D). PF inhibits the migration of preosteoclasts Because downregulation of podosome-related proteins and defects in the formation of protrusions affect cell migration, we Fig. 3. PF inhibits podosome-related protein expression and protrusion in preosteoclasts. (A) Changes in the expression of podosomerelated proteins during RANKL-induced osteoclast differentiation were evaluated by Western blot analysis. (B) Protein expression levels were evaluated in preosteoclasts derived from BMMs after treatment with PF for 1 day. (C) BMMs differentiated into preosteoclasts in the absence or presence of PF The preosteoclasts were subsequently fixed, stained, and visualized using Cellomics ArrayScan. The merged images show nuclei (blue), vinculin (green), and p-pak4 (red). Data are representative of at least three experiments CHOI ET AL. Journal of Bone and Mineral Research

8 Fig. 4. NFATc1 overexpression can overcome the antiosteoclastogenic effects of PF (A) BMMs infected with pmx-control or pmx-ca-nfatc1 (CA-NFATc1) were cultured with M-CSF (30 ng/ml) and RANKL (10 ng/ml) for 3 days in the presence or absence of PF (5 nm). TRAP þ -MNCs were visualized by TRAP staining. (B) TRAP þ -MNCs were counted (n ¼ 3), and (C) the TRAP activity was measured (n ¼ 3). **p < 0.01, ***p < versus the controls. (D) The effect of NFATc1 overexpression on the expression of other proteins was evaluated by Western blot analysis. Data are representative of at least three experiments. evaluated the effect of PF on the migration of preosteoclasts using two migration assay systems (Fig. 5A D). Results of the Boyden chamber migration assay and the woundhealing assay revealed that PF significantly inhibited the migration of preosteoclasts in a dose-dependent manner (Fig. 5A D, Supporting Fig. 7, Supporting Videos 1 5). The detailed statistical analysis results are shown in Supporting Table 3. Furthermore, we evaluated whether the inhibitory effect of PF on the migration of preosteoclasts is dependent on cell density (Fig. 5E). When the migration assay was repeated, plating low ( cells/well), medium (1 or cells/ well), and high numbers of osteoclast precursors ( cells/ well), PF significantly inhibited the migration of osteoclast precursors when cells were seeded at low and medium densities. However, at high cell density, it did not inhibit the migration, suggesting that osteoclast formation could not be affected at high density of osteoclast precursors because cells were already close to each other and did not need migration to fulfill fusion. PF prevents LPS-induced bone erosion in vivo The antiresorptive activity of PF was evaluated in vivo in the LPS-induced mouse bone erosion model. As shown in Fig. 6A, mct indicated that trabecular bone in the metaphyseal region of the femur was eroded by LPS treatment and that administration of PF dramatically prevented LPSmediated trabecular bone loss. Specifically, LPS-induced changes in BMD, BV/TV, Tb.N, and Tb.Sp were substantially prevented by PF (Fig. 6B). In addition, there were no injuries of vital organ and no changes in serum markers by PF treatment (Supporting Fig. 8). Histological assessments also revealed that LPS-induced decrease of trabecular bone volumes and increase of osteoclast surface in the epiphyseal region were notably restored by PF (Fig. 6C, D). Also, the immunohistochemistry analysis revealed that LPS-mediated induction of p-pak was attenuated by PF (Supporting Fig. 9). PF inhibits the RANKL-induced differentiation of human osteoclasts Finally, the antiosteoclastogenic activity of nanomolar concentrations of PF was evaluated using hpbmcs and hbmcs. As shown in Fig. 7A and B, recombinant human RANKL (rhrankl) strongly induced the formation of TRAP þ -MNCs derived from hpbmcs, and PF significantly inhibited this induction in a dose-dependent manner. In addition, the rhrankl-induced formation of TRAP þ -MNCs derived from hbmcs was significantly inhibited by PF Discussion PAK4 controls cell migration and podosome formation, suggesting its relevance in osteoclast differentiation. Here we observed high levels of PAK4 in BMMs and the gradual induction of PAK4 phosphorylation during RANKL-mediated osteoclast differentiation. Our results further suggested that PAK4 kinase activity may be essential for osteoclast differentiation, because PAK4 kinase mutants and the PAK4 inhibitor PF had Journal of Bone and Mineral Research REPOSITIONING POTENTIAL OF PAK4 TO OSTEOCLASTIC BONE RESORPTION 1501

9 Fig. 5. PF suppresses preosteoclast invasion and migration. The effects of PF on the (A) invasion and (B E) migration of preosteoclasts were measured using a Boyden chamber and a wound healing assay, respectively. (A) Invaded cells were counted (n ¼ 3), and (B) the wound edge (indicated with white) and the relative wound density were analyzed at 24 hours (C) or for periods of time (D) (n ¼ 6). (E) The cell density-dependent effects of PF on the migration of preosteoclasts were measured using a wound healing assay. The relative wound density was analyzed at 24 hours (n ¼ 6). Data are representative of at least three experiments. *p < 0.05, **p < 0.01, and ***p < versus the controls CHOI ET AL. Journal of Bone and Mineral Research

10 Fig. 6. PF prevents LPS-induced in vivo bone erosion. The effect of PF on LPS-induced bone erosion was investigated in mice. (A) mct analysis-based 3D rendering shows transverse and longitudinal images, and (B) BMD, BV/TV, Tb.N, and Tb.Sp femur measurements were performed using CTAn software (n ¼ 5). ##p < 0.01 (versus the controls); *p < 0.05 (versus the LPS-treated group). (C) All mice were euthanized 8 days after the initial LPS injection, and femurs were dissected, fixed, decalcified, and cut into sections. Sections were stained with H&E and other sections were stained with TRAP solution. Sections were also counterstained with methylene blue. Stained bones were photographed under a light microscope (40). (D) Osteoclast surface per bone surface (Oc.S/BS) was analyzed in TRAP-stained sections (n ¼ 5). ##p < 0.01 (versus controls); *p < 0.05 and **p < 0.01 (versus the LPStreated group). One representative result obtained from three independent experiments yielding similar results was shown. Journal of Bone and Mineral Research REPOSITIONING POTENTIAL OF PAK4 TO OSTEOCLASTIC BONE RESORPTION 1503

11 Fig. 7. PF has antiosteoclastogenic activity in human cells. The effects of PF on hpbmcs and hbmc osteoclast differentiation were evaluated by (A) TRAP staining and by counting the number of TRAP þ -MNCs (B). Data are representative of at least three experiments (n ¼ 4). *p < 0.05 and ***p < versus the RANKL-treated group. antiosteoclastogenic activity at nanomolar concentrations. Specifically, PF strongly inhibited the fusion of preosteoclasts; it also inhibited PAK4 phosphorylation and the expression of fusion/maturation-related genes that encode NFATc1, dendrite cell-specific transmembrane protein (DC- STAMP), cathepsin K, and the d2 isoform of vacuolar (H þ ) ATPase V 0 domain (ATP6vOd2). These results suggested that the pharmacological inhibition of PAK4 by PF inhibited the fusion of preosteoclasts in the late stage of osteoclast differentiation via downregulation of NFATc1 expression and that this subsequently affected the transcription of osteoclast differentiation-related proteins. Osteoclast differentiation is a multistep progressive process that involves cell proliferation, commitment, migration, fusion, and maturation. (25,26) Monocyte/macrophage precursors derived from hematopoietic stem cells in the bone marrow become preosteoclasts that then migrate and fuse with each other to form giant multinucleated osteoclasts. NFATc1 is a master regulator of RANKL-induced osteoclast differentiation; in the absence of RANKL, overexpression of NFATc1 induces the differentiation of osteoclast precursors into osteoclasts. In contrast, NFATc1-deficient embryonic stem cells fail to differentiate into osteoclasts, even in the presence of RANKL. (27) To elicit the osteoclast phenotype, NFATc1 binds directly to the promoter regions and induces the expression of its target genes. (28) DC-STAMP and ATP6vOd2 contain multiple NFATc1 binding sites in their promoter regions, (29) and the proteins encoded by both of these genes are reported to play roles in osteoclast fusion. (30,31) NFATc1 also regulates the activity of the b 3 integrin and cathepsin K promoters. (32,33) Interestingly, accumulation of a v b 3 integrin on the cell surface is triggered by the aggregation of mononuclear preosteoclasts and correlates with the spreading of macrophages that precedes fusion. (34) This suggests the involvement of a v b 3 integrin in the fusion of preosteoclasts. Cell-cell fusion is essential for the formation and maturation of multinucleated osteoclasts, and this process involves unique cell adhesion structures called podosome clusters or actin rings. Podosomes consist of a core of F-actin bundles surrounded by a rosette-like structure containing a v b 3 integrin and actin-binding proteins such as vinculin, cofilin, and cortactin. The regulation of these molecules is tightly associated with the fusion of preosteoclasts and with the bone resorption activity of mature osteoclasts due to their effects on the reorganization of actin assembly. Vinculin, which is an osteoclast microfilament protein, colocalizes with b 3 integrin around the actin core of the podosome in human osteoclasts. (35) Cofilin, an actin-severing protein, also regulates podosome organization, and its dephosphorylation and activation is increased during RANKL-induced osteoclast differentiation. (36) Cortactin, an actin-associated protein, is enriched in the core of podosomes and, when cortactin is depleted, cells fail to form actin-based sealing rings and resorb the bone matrix. (37) Interestingly, the activity of PAK4 regulates podosome formation in macrophages, (15) and in this study we found that the pharmacological inhibition of PAK4 by PF inhibited the RANKL-induced expression and/or activation of podosome-related proteins. Consistent with this, the RANKL-induced protrusions in preosteoclasts were inhibited by PF These results suggest that PF may inhibit the formation of protrusions in preosteoclasts by downregulating podosome-related proteins such as vinculin. Moreover, the restoring effect of NFATc1 overexpression on the PF mediated inhibition of osteoclast differentiation further suggests that the antiosteoclastogenic activity of PF is mediated by inhibition of the PAK4-NFATc1-vinculin signaling axis. Altering the PAK4 activity changed the expression 1504 CHOI ET AL. Journal of Bone and Mineral Research

12 levels of NFATc1 and vinculin proteins, and vinculin expression was strongly induced by NFATc1 overexpression and maintained even in the presence of PF In addition to their roles in cell fusion and podosome formation, vinculin, cortactin, cofilin, and related proteins also play crucial roles in cell migration. Podosomes contain actin remodeling proteins and associated signaling components that are responsible for actin assembly and disassembly. (38,39) In osteoclasts, changes in podosome assembly and disassembly allow cell migration, adhesion, and bone resorption. The functional role of the a v b 3 integrin in osteoclast migration is reflected by its localization to podosomes at the leading edges of migrating osteoclasts and by the inhibitory effect of a v b 3 integrin antagonist on osteoclast migration and bone resorption in vivo. (40,41) In our study, PF dose-dependently inhibited the migration of preosteoclasts, suggesting that the PF mediated inhibition of preosteoclast migration suppressed their fusion. As noted above, PAK4 regulates cell migration and podosome formation. (15,18) The in vitro antiosteoclastogenic activity of PF prompted us to investigate its in vivo efficacy and possible applications in humans. In addition to RANKL, inflammatory LPS and LPS-dependent proinflammatory cytokines, such as tumor necrosis factor (TNF)-a, are responsible for bone resorption by inducing the fusion of mononuclear cells to generate multinucleated osteoclasts. (42 45) Conditioned medium from LPStreated BMMs and TNF-a enhance the migration of osteoclast precursors, (46,47) and LPS itself mediates podosome disassembly. (48) Although the relevance of PAK to inflammation remains unknown, endogenous PAK4 is required for the full activation of TNF-a induced prosurvival pathways. In the LPS-induced bone erosion animal model used in this study, PF showed in vivo antiosteoclastogenic activity. Notably, even the lower dosages of PF (0.5 to 1 mg/kg i.p. daily) significantly protected mice against LPS-mediated bone erosion, suggesting that it has beneficial in vivo effects in inhibiting inflammationrelated bone loss. Furthermore, even at picomolar to nanomolar concentrations, the RANKL-induced formation of TRAP þ -MNCs from both hpbmcs and hbmcs was significantly inhibited by PF in a dose-dependent manner, suggesting that it has potential as a human therapeutic. There are osteoclast precursor cells that express RANK in human peripheral blood, and PBMCs isolated from postmenopausal osteoporotic patients can spontaneously differentiate into osteoclasts. (49) In addition, the ovariectomized model was not carried out in this study because PF with the undesirable pharmacokinetic characteristics and the lack of an observed dose-response relationship in the phase I might not be enough for its oral administration. Therefore, we are trying to develop novel oral available PAK4 inhibitors with antiresorptive activity and/or anticancer activity. (50) The upstream molecules to activate PAK4 during osteoclast differentiation might be identified in a further study. Basically, small GTPases including RhoA, Rac, and Cdc42 have been shown to regulate osteoclast function and bone resorption as upstream signaling molecules of PAK. (51) Constitutively active Rho transduction stimulated podosome assembly, osteoclast motility, and bone resorption, (52) but Rac1 reduction in osteoclasts of its knockout mice caused severe osteopetrosis. (53) Cdc42 has been shown to be required for multiple M-CSF induced and RANKL-induced signaling for osteoclast differentiation. (54) In addition, Vav3, a Rho family GTP exchange factor, is essential for stimulating osteoclast activation and maintaining bone density in vivo; its deficient osteoclasts exhibit defective actin cytoskeleton organization and resorptive activity. (55) The functional relationship between small GTPases and PAK4 during osteoclast differentiation could be helpful to understand the osteoclastogenic action of PAK4. In summary, this is the first study to show that PAK4 may be relevant in osteoclast-related diseases because of the antiosteoclastogenic effects of PAK4 inhibition. In particular, pharmacological inhibition of PAK4 by its inhibitor PF at nanomolar concentrations blocked the migration/fusion of preosteoclasts and downregulated NFATc1. Moreover, PF showed in vitro antiosteoclastogenic activity in human cells at picomolar concentration and in vivo antiresorptive activity in an LPS-induced bone erosion animal model when administrated at doses of 0.5 to 1 mg/kg i.p. daily. Although the potential of PAK4 as a novel therapeutic for osteoclast-related diseases must be confirmed by a more detailed clinical proof-ofconcept study, the results presented here suggest that the clinical indications of PAK4 might be expanded to include osteoclast-related diseases, including osteoporosis, rheumatoid arthritis, and cancer bone metastasis. Disclosures All authors state that they have no conflicts of interest. Acknowledgments This work was supported by the project grant (SI-1404) of Korea Research Institute of Chemical Technology. Authors roles: SWC and JTY contributed equally to this work. SWC contributed to all experiments and the manuscript preparation. JTY performed the in vivo study. BJR performed the migration/invasion assay. KJK contributed to the realtime PCR and Western blot experiments, and performed the retrovirus-based functional study. SHM contributed to the cloning of PAK4 constructs and the osteoblast differentiation study. HL synthesized PF MSL and SYL isolated hbmcs, and prepared the part of the manuscript. JCH performed the immunofluorescence-based study. SJP supervised the in vivo research. SHK designed and supervised all of experiments, and wrote and finalized the manuscript. References 1. Coles LD, Cloyd JC. The role of academic institutions in the development of drugs for rare and neglected diseases. Clin Pharmacol Ther. 2012;92(2): Li YY, Jones SJ. Drug repositioning for personalized medicine. Genome Med. 2012;4(3): Gupta SC, Sung B, Prasad S, Webb LJ, Aggarwal BB. Cancer drug discovery by repurposing: teaching new tricks to old dogs. Trends Pharmacol Sci. 2013;34(9): Takayanagi H. Osteoimmunology and the effects of the immune system on bone. 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