Ca2+-Dependent Regulation of NFATc1 via KCa3.1 in Inflammatory Osteoclastogenesis Eva M. Grössinger, Mincheol Kang, Laura Bouchareychas, Ritu Sarin, Dominik R. Haudenschild, Laura N. Borodinsky This information is current as and Iannis E. Adamopoulos of September 26, 2021. J Immunol published online 15 December 2017 http://www.jimmunol.org/content/early/2017/12/15/jimmun ol.1701170 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 15, 2017, doi:10.4049/jimmunol.1701170 The Journal of Immunology

Ca2+-Dependent Regulation of NFATc1 via KCa3.1 in Inflammatory Osteoclastogenesis

Eva M. Gro¨ssinger,* Mincheol Kang,* Laura Bouchareychas,* Ritu Sarin,* Dominik R. Haudenschild,† Laura N. Borodinsky,‡,x and Iannis E. Adamopoulos*,x

In inflammatory arthritis, the dysregulation of osteoclast activity by proinflammatory cytokines, including TNF, interferes with bone remodeling during inflammation through Ca2+-dependent mechanisms causing pathological bone loss. Ca2+-dependent CREB/c-fos activation via Ca2+-calmodulin kinase IV (CaMKIV) induces transcriptional regulation of osteoclast-specific via NFATc1, which facilitate bone resorption. In leukocytes, Ca2+ regulation of NFAT-dependent expression oftentimes involves the activity of the Ca2+-activated K+ channel KCa3.1. In this study, we evaluate KCa3.1 as a modulator of Ca2+-induced NFAT-dependent osteoclast differentiation in inflammatory bone loss. Microarray analysis of receptor activator of NF-kB ligand

(RANKL)-activated murine bone marrow macrophage (BMM) cultures revealed unique upregulation of KCa3.1 during osteo- Downloaded from clastogenesis. The expression of KCa3.1 in vivo was confirmed by immunofluorescence staining on multinucleated cells at the bone surface of inflamed mouse joints. Experiments on in vitro BMM cultures revealed that KCa3.12/2 and TRAM-34 treatment significantly reduced the expression of osteoclast-specific genes (p < 0.05) alongside decreased osteoclast formation (p < 0.0001) in inflammatory (RANKL+TNF) and noninflammatory (RANKL) conditions. In particular, live cell Ca2+ imaging and Western blot analysis showed that TRAM-34 pretreatment decreased transient RANKL-induced Ca2+ amplitudes in BMMs by ∼50% 2 2 (p < 0.0001) and prevented phosphorylation of CaMKIV. KCa3.1 / reduced RANKL+/2TNF-stimulated phosphorylation of http://www.jimmunol.org/ CREB and expression of c-fos in BMMs (p < 0.01), culminating in decreased NFATc1 expression and transcriptional activity (p < 0.01). These data indicate that KCa3.1 regulates Ca2+-dependent NFATc1 expression via CaMKIV/CREB during inflammatory osteoclastogenesis in the presence of TNF, corroborating its role as a target candidate for the treatment of bone erosion in inflammatory arthritis. The Journal of Immunology, 2018, 200: 000–000.

hysiological bone remodeling is a balanced process creating an isolated environment on the bone surface into which maintained by the activity of bone-resorbing osteoclasts enzymatic vesicles that contain lytic enzymes including tartrate- and bone-forming osteoblasts. Osteoclasts differentiate resistant acid phosphatase (TRAP; Acp5), matrix metalloproteinase-9 P by guest on September 26, 2021 from myeloid precursor cells into multinucleated giant cells under (MMP9; Mmp9), and cathepsin K (CTSK; Ctsk) can be released to the influence of M-CSF and receptor activator of NF-kB ligand facilitate bone resorption (1–6). The transcriptional regulation of (RANKL) (1–5). Terminally differentiated osteoclasts are charac- osteoclastogenesis by RANKL is initiated by RANK/RANKL terized by the formation of sealing zones of F-actin (F-actin rings), binding, which induces Ca2+-independent NF-kB signaling, as well as c-fos/AP-1, both resulting in the expression of NFATc1, the master regulator of osteoclastogenesis (7–10). Simultaneously, *Division of Rheumatology, Allergy, and Clinical Immunology, Department of In- 2+ ternal Medicine, University of California Davis, Davis, CA 95616; †Center for Mus- RANKL induces c-fos/AP-1 upregulation through Ca -dependent culoskeletal Health, University of California Davis, Sacramento, CA 95817; signaling cascades involving phospholipase Cg (11–13). The ‡Department of Physiology and Membrane Biology, University of California Davis, 2+ 2+/ x increase in intracellular Ca activates the Ca calmodulin- Davis, CA 95616; and Institute for Pediatric Regenerative Medicine, Shriners Hos- 2+ pital for Children – Northern California, Sacramento, CA 95817 dependent Ca -calmodulin kinase IV (CaMKIV), capable of ORCIDs: 0000-0003-4962-9750 (L.B.); 0000-0001-9947-9864 (D.R.H.). inducing transcriptional upregulation of c-fos/AP-1 and Nfatc1 Received for publication August 15, 2017. Accepted for publication November 2, through phosphorylated CREB (pCREB) (9, 14, 15). Subse- 2017. quently, NFATc1 undergoes autoamplification by binding to its 2+ This work was supported by Austrian Science Fund FWF Project J3715-B26 to own promoter region (16). Besides CaM kinases, the Ca / E.M.G., National Institutes of Health/National Institute of Neurological Disorders calmodulin-dependent phosphatase calcineurin (CaN) is also ac- and Stroke Grant R01NS073055 and National Science Foundation 1120796 to 2+ L.N.B., National Institutes of Health/National Institute of Arthritis and Musculoskeletal tivated by increased intracellular Ca concentrations (17, 18). and Skin Diseases Grant R01AR062173, and a National Psoriasis Foundation Transla- Activated CaN dephosphorylates NFATc1 enabling its nuclear tional Research grant to I.E.A. translocation. Next, a transcriptional complex including NFATc1, Address correspondence and reprint requests to Dr. Iannis E. Adamopoulos, University c-fos/AP-1, and pCREB trigger the transcription of osteoclast- of California Davis, Division of Rheumatology, Allergy and Clinical Immunology and Institute for Pediatric Regenerative Medicine, Shriners Hospital for Children – Northern specific genes (13, 19). During pathological conditions, as in in- California, 2425 Stockton Boulevard, Sacramento, CA 95817. E-mail address: flammatory arthritis, alternative stimuli can facilitate osteoclast [email protected] formation in addition to the classic RANKL pathway (20–25). The online version of this article contains supplemental material. Proinflammatory cytokines such as TNF promote osteoclast Abbreviations used in this article: BMM, bone marrow macrophage; CaMKIV, Ca2+-calmodulin kinase IV; CaN, calcineurin; CRAC, Ca2+ release-activated calcium; formation and activity in rodents and humans (24, 26). TNF is CTSK, cathepsin K; IF, immunofluorescence; MMP9, matrix metalloproteinase-9; released by several types of T cells as well as by synovial mac- pCREB, phosphorylated CREB; qPCR, quantitative PCR; RANKL, receptor activator rophages under inflammatory joint conditions, and is involved in of NF-kB ligand; TRAP, tartrate-resistant acid phosphatase; WT, wild type. promoting NF-kB signaling as well as Ca2+-dependent pathways Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 in osteoclast precursor cells (24). Hence, both RANKL- and

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701170 2 KCa3.1 REGULATES NFATc1 IN INFLAMMATORY OSTEOCLASTOGENESIS

TNF-dependent osteoclast differentiation pathways are critically IF and H&E staining dependent on Ca2+ signals (13, 14, 24). 2+ Native cells were fixed with 4% paraformaldehyde solution and blocked In leukocytes, Ca -dependent NFAT signaling through CaN is with 5% donkey serum for 1 h at room temperature. Primary KCa3.1 Ab was modulated by K+ channel activity, which has been thoroughly used (1:300) and conjugated to secondary donkey IgG affinity purified examined in the past three decades (27). During phospholipase fluorescently labeled Abs (1:500). As controls for unspecific binding, Cg-induced store-operated Ca2+ entry through plasma membrane secondary Abs were used without primary. Samples were embedded using 2+ 2+ Vectashield mounting medium for IF (Vector Laboratories), containing Ca channels such as Ca release-activated calcium (CRAC) DAPI. For staining of mouse joints, whole ankle and knee joints were fixed + channels, K channels repolarize the plasma membrane by cre- in 10% formalin decalcified in 10% EDTA and embedded in paraffin. Serial ating a positive outward current and thereby stabilize the driving sections (4 mm) were rehydrated in alcohol gradient and stained with H&E. force for Ca2+ influx, a prerequisite for sustained activation of For IF, Ag was retrieved using 0.6 U/ml Proteinase-K solutions. Subse- quent Ab-labeling and mounting for IF was performed as with native cells. CaN-dependent subsequent through NFAT + H&E imaging was obtained by Olympus BX61 confocal microscope (12, 27–30). However, the dependence of CaMK pathways on K (Olympus) and analyzed with cellSens Dimension software (Olympus). IF channel activity during leukocyte differentiation and its role in imaging was performed with a Nikon A1 confocal microscope (Nikon NFAT expression is much less understood at this time (31). Instruments) with 2003 or 6003 magnification. Images within one ex- Recent studies have highlighted the importance of the Ca2+-dependent periment were recorded under identical parameters to ensure comparability and analyzed using ImageJ 1.46. K+ channel KCa3.1 (Kcnn4) as a key regulator during osteoclast formation (32), and suggested that the mechanism of action of Microarray analysis Ibandronate, a commonly used drug to treat postmenopausal os- Total RNA was amplified and purified using an Illumina RNA amplification teoporosis, involves the inhibition of KCa3.1 currents (33). kit (Ambion) to yield biotinylated cRNA, and 1.5 mg of cRNA samples Downloaded from In the current study we characterize the involvement of the were hybridized to Illumina WG-6 v2.0 chips according to the manufac- predominant K+ channel KCa3.1 in CaMK/CREB/c-fos–dependent turer’s instructions (Illumina). Arrays were scanned by Illumina iScan. 2+ Raw data were extracted using GenomeStudio (Illumina). Probe signal Ca signaling and NFATc1 regulation during inflammatory values were transformed by mean normalization and logarithm, and compared with noninflammatory osteoclast differentiation in the then illustrated by a heat map. Data are deposited in the National Center presence and absence of TNF using in vitro osteoclast assays, as for Biotechnology Information Gene Expression Omnibus under acces- 2+ well as live cell Ca imaging. sion number GSE86998 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? http://www.jimmunol.org/ acc=GSE86998). Materials and Methods AlamarBlue assay Reagents and Abs AlamarBlue cell viability and proliferation assay was performed according to the manufacturer’s instructions (Invitrogen). In brief, cells were culti- Cells were cultured in aMEM containing 2 mM L-glutamine (12561), 10% heat-inactivated FBS, 100 IU/ml penicillin, and 100 IU/ml streptomycin vated in 96-well plates. All samples were plated in triplets. AlamarBlue (Life Technologies). Quantitative PCR (qPCR) primers were either reagent was added to each well and incubated on 37˚C for 7 h before fluorescence detection with an excitation at 570 nm and emission at designed or used as previously described and tested for their distinctive 590 nm. product sizes (Integrated DNA Technologies) (34). CMG-1412 media were generated as previously described (35). Immunofluorescence (IF) Abs and Calcium measurements by guest on September 26, 2021 reagents are: anti-KCa3.1 (P4997) (Sigma-Aldrich), anti-CD68 (FA-11) (BioLegend) (36), phalloidin-FITC (Sigma-Aldrich), goat anti-rabbit AF-594 Bone marrow macrophages (BMMs) were cultured in M-CSF (25 ng/ml) and goat anti-rat AF488 (Life Technologies), and goat serum (Gemini Bio for4d.PriortoCa2+ measurements, cells were starved of M-CSF for Products). Recombinant cytokines (mM-CSF, mTNF, and mRANKL) were 6–8 h. Directly before imaging, cells were labeled with 2.5 mM Fluo-4 purchased from R&D Systems; inhibitors: TRAM-34, KN-93, cyclospor- AM. Cells were observed for baseline Ca2+ for 5 min before addition of ine A (CsA) (Sigma-Aldrich); Fluo-4-AM (Life Technologies); Western 100 ng/ml RANKL. For KCa3.1 inhibition, cells were preincubated with blot Abs: anti-CREB (06-863) and anti–phospho-CREB (Ser133) (06-519) 3 mM TRAM-34 or DMSO for 15 min at 37˚C prior to Ca2+ measure- (Millipore) (14), anti-CaMKIV (H-5) (Santa Cruz Technologies) (37), ments. Fluo-4 AM intensity was measured and tracked in 1 s intervals anti–phospho-CaMKIV (Thr196, Thr200) (PA5-37504) (Thermo Fisher for 15 min using a Nikon Eclipse FN1 Swept-Field Confocal micro- Scientific), anti-NFATc1 (7A6) (Thermo Fisher Scientific) (14), anti–b-actin scope with 2003 magnification and NIS-Elements BR software (Nikon (13E5) (Cell Signaling Technology) Instruments). Animals RNA isolation and qPCR All animal protocols and procedures are in accordance with the Declaration Total RNA isolation was performed using an RNeasy Mini RNA iso- of Helsinki and were approved by the UC Davis Animal Care and Use lation kit (Qiagen) according to the manufacturer’s instructions. RNA Committee (Institutional Animal Care and Use Committee). C57BL/6J and yield was measured by photospectrometry. Briefly, 500 ng of total RNA C57BL/6J Kcnn42/2 mice, originally purchased from The Jackson Lab- was used for cDNA synthesis using oligo(dT)15 primers and an oratory, were subsequently bred in the UC Davis animal facility under Omniscript Reverse Transcription cDNA synthesis kit following the standardized breeding conditions. For in vitro experiments, mice were manufacturer’s recommendations (Qiagen). qPCR was performed us- sacrificed by carbon dioxide exposure. Arthritis was induced using the ing QuantiTect SYBR Green PCR kit following the manufacturer’s IL-23 gene transfer model as previously described (38). instructions (Qiagen); all cDNAs were standardized to b2M mRNA expression. Osteoclast cultures and characterization Trans-AM NFATc1 transcription factor binding assay Whole bone marrow was extracted from tibiae and femora of 8–12 wk old wild-type (WT) C57BL/6J and Kcnn42/2 mice, as previously described Transcription factor binding assay was performed according to the man- (39). Cells were plated in culture medium containing CMG-1412 cell line ufacturer’s instructions (Active Motive). In brief, BMMs were cultivated in medium 1:20 (v/v) in aMEM for 4 d until 30–40% confluence to generate 10 cm culture dishes and stimulated with RANKL (30 ng/ml) alone or in macrophages. To generate osteoclasts, the culture medium was then sup- combination with TNF (10 ng/ml) as described earlier. After 36 h, nuclear plemented with soluble recombinant mRANKL (30 ng/ml) for an addi- lysates were extracted using Nuclear Extract kit (Active Motive). Final tional 3 d. For inflammatory conditions, soluble recombinant mTNF was protein concentrations were determined using a BCA protein assay kit added at 10 ng/ml. Cytochemical staining was performed using an Acid (Pierce). Protein lysates were then incubated in an oligonucleotide pre- Phosphatase, Leukocyte (TRAP) Kit according to the manufacturer’s in- coated plate and detected with primary NFATc1 and secondary structions (Sigma-Aldrich). For F-actin ring staining, cells grown on cov- HRP-labeled Abs. Absorbance was measured at 450 nm with a reference erslips were fixed with 4% paraformaldehyde solution and permeabilized wavelength at 655 nm. Assay specificity was guaranteed by competitive using 0.5% Triton X-100 before incubation with Phalloidin-FITC (1:100) for binding assays using WT and control mutated consensus sequences for 1 h at room temperature. NFATc1. The Journal of Immunology 3

Western blot analysis ***p , 0.001, ****p , 0.0001; calcium measurements: for population analysis, the number of responding cells was normalized to the total Time-dependent phosphorylation experiments were performed in total cell number of cells per frame; for assessment of the response magnitude, the lysates of BMMs and osteoclasts, respectively. For CREB phosphorylation, maximum response after stimulation was normalized to the maximum cells were cultivated in full culture medium and stimulated with 30 ng/ml response baseline (DRA/DBL). RANKL or RANKL in combination with 10 ng/ml TNF for indicated times before cell lysates were collected. For CaMKIV phosphorylation, cells were Results starved for 6–8 h before stimulation with 100 ng/ml RANKL. BMMs and osteoclasts were lysed in radioimmunoprecipitation buffer (Cell Signaling KCa3.1 is highly expressed during osteoclastogenesis Technology) including phosphatase inhibitors (Pierce) and Mini EDTA- To investigate the regulation of K+ channel expression during free Protease Inhibitor Cocktail (Roche). Insoluble material was removed osteoclast differentiation, we performed RNA microarray analysis by centrifugation at 13,000 rpm for 20 min at 4˚C. Final protein concen- trations were determined using a BCA protein assay kit (Pierce). Elec- on isolated mouse BMMs cultivated from murine M-CSF–stimulated trophoresis was performed using four ∼ 15% Mini-PROTEAN TGX bone marrow cells before and after RANKL-induced osteoclast Precast Gels (Bio-Rad). Blots were subsequently transferred to polyvi- differentiation (Supplemental Fig. 1). A comparison of a number of nylidene difluoride membranes and unspecific binding was blocked with selected K+ channels, previously described in macrophage or osteo- 5% BSA in TBST. Immunoreactive bands were detected with the Odyssey clast biology (32, 40), identified the upregulation of only one Ca2+ Infrared Imaging System (LI-COR Biosciences). All specific bands were + normalized to b-actin signals. -activated K channel, KCa3.1 (Kcnn4), during RANKL-induced osteoclastogenesis (Supplemental Fig. 1A, 1D). Statistical analysis We also investigated the expression of KCa3.1 in murine in- Statistical analysis and graphical representation was performed using flammatory arthritis in vivo (38) and confirmed that multinucleated GraphPad Prism Version 6.03 and Windows Excel 2010. Following a cells located at the bone surface of inflamed joints (Fig. 1A) Downloaded from Gaussian distribution (D’Agostino and Pearson omnibus normality test), coexpressed KCa3.1 and CD68 (Fig. 1B) (41). Similarly, qPCR p values were calculated for a confidence interval of 95% by paired or unpaired Student t test. When calculating p values for normalized data, analysis of BMMs stimulated with RANKL in vitro revealed up- one-sample t test for a hypothetical value of 1.0 was assumed. In case of regulation of KCa3.1 mRNA (Kcnn4) during RANKL-induced nonparametric data either Mann–Whitney U or Wilcoxon-ranked pairs test osteoclastogenesis, whereas the presence of TNF in addition to were used. For multiparameter experiments, either one-way ANOVA RANKL had no significant influence on Kcnn4 expression

preceding post hoc Holm–Sidak multiple comparison test for parametric http://www.jimmunol.org/ data or Kruskal–Wallis followed by Dunn multiple comparisons test were (Fig. 1C). Interestingly, the expression of the voltage-gated po- performed for a confidence interval of 95%, unless otherwise indicated. tassium channel Kv1.3 (Kcna3), which has previously been Bar graphs show means with SEM; n.s. = p . 0.05, *p , 0.05, **p , 0.01, demonstrated to play a role in proinflammatory macrophages and by guest on September 26, 2021

FIGURE 1. KCa3.1 is present on multinucleated cells in inflammatory arthritis. (A) H&E staining of paraffin sections obtained from inflamed joints of arthritic mice 11 d after IL-23 minicircle injection showing a multinucleated cell or osteoclast (OC) at the bone surface adjacent to inflamed periosteum (scale bar, 20 mm). (B) IF staining of a multinucleated cell at the bone surface showing coexpression of CD68 (green) and KCa3.1 (red) with DAPI (blue) (scale bar, 20 mm). (C) qPCR analysis showing normalized expression of active (Kcnn4 a) and dominant-negative (Kcnn4 b) splice variants of the KCa3.1 gene (Kcnn4); Kcnn4 a/b primer detecting both transcripts. One-way ANOVA followed by post hoc multiple comparison test was performed (n =4,p , 0.0001); shown are mean 6 SEM. *p , 0.05, **p , 0.01. (D) IF staining for KCa3.1 (red), actin (green), and DAPI (blue) in cultured mouse BMMs, unstimulated (control), and stimulated in indicated conditions (n = 3, scale bar, 100 mm). 4 KCa3.1 REGULATES NFATc1 IN INFLAMMATORY OSTEOCLASTOGENESIS microglia differentiation, was found to be significantly underrep- a mean reduction of 47.8% in normalized Ca2+ transient ampli- resented in macrophages and osteoclasts (Supplemental Fig. 1E) tudes in TRAM-34 compared with control pretreated cells (42). By using splice variant-specific primers, we also detected the (Fig. 3C, right), suggesting a diminished Ca2+ influx caused by the presence of a dominant-negative transcript of Kcnn4 (Kcnn4 b) lack of KCa3.1-dependent membrane repolarization. next to the active variant (Kcnn4 a) in macrophages and osteo- Changes in Ca2+ signaling can lead to phosphorylation and clasts, as suggested by a previous study in lymphocytes (34). IF activation of Ca2+-sensitive enzymes such as CaN and CaMKIV staining of KCa3.1 confirmed protein expression in cultures (14, 18). We also found that the pharmacological inhibition of stimulated with RANKL or TNF alone and the combination of both CaMKIV by KN-93 and CaN by cyclosporine A significantly both in multinucleated cells exhibiting F-actin ring structures decreased osteoclast formation and expression of osteoclast- (Fig. 1D). specific genes during inflammatory conditions using RANKL in These results indicate that KCa3.1 is expressed during physi- combination with TNF (Supplemental Fig. 3A–C). This indicated ological and inflammatory osteoclastogenesis. that activity of either transducer is essential for osteoclastogenesis during inflammation. To investigate a direct interference of altered KCa3.1 function is required for osteoclast formation and gene Ca2+ signaling through KCa3.1 activity with CaMKIV, we stim- expression in physiological and inflammatory osteoclast ulated BMMs with RANKL and show that an increase in CaMKIV differentiation phosphorylation was prevented by inhibiting KCa3.1 with TRAM- To further understand the role of KCa3.1 in osteoclastogenesis 34 or K+ efflux through increasing the K+ concentration in the during inflammation, we stimulated BMMs from WT and Kcnn42/2 culture medium (Supplemental Fig. 3D). These data suggest that mice with RANKL and RANKL in combination with TNF. Al- both CaN and CaMKIV are likely to be involved in effects derived Downloaded from though visibly smaller in size, as previously described by Kang from altered Ca2+ signaling through KCa3.1 inhibition during et al. (32), cultures from Kcnn42/2 cells compared with WT physiological and inflammatory osteoclast formation. revealed similar numbers of TRAP+, multinucleated cells in the KCa3.1 modulates protein expression and activity of NFATc1 presence of RANKL when assessed with a threshold of $3 nuclei through CREB and c-fos (Fig. 2A, 2B). However, combining RANKL and TNF stimulation

reduced those numbers significantly by 40.8%. Pharmacological CaMKIV phosphorylates and thereby modulates transcriptional http://www.jimmunol.org/ inhibition of KCa3.1, using the specific KCa3.1 inhibitor TRAM- activity of CREB during RANKL-induced osteoclast differentiation 34, revealed a decrease in multinucleated cells regardless of the (14). We next aimed to understand the consequences of KCa3.1 presence (38.9% reduction) or absence (29.4% reduction) of TNF deletion on CREB phosphorylation in inflammatory (RANKL (Fig. 2A, 2B), suggesting potential compensation of KCa3.1 +TNF) compared with noninflammatory (RANKL) osteoclasto- 2/2 functions in Kcnn42/2 cells that partially rescues the osteoclast genesis. Analysis of BMMs from WT and Kcnn4 mice stimu- formation phenotype when stimulated with RANKL alone. The lated with RANKL and RANKL in combination with TNF for 36 h, inhibition of KCa3.1 by TRAM-34 was found to be concentration- a time period assessed to yield maximal pCREB in WT cells (data dependent (Supplemental Fig. 2A) and cell viability and prolif- not shown), revealed that both RANKL- and RANKL-TNF–co- 2/2 eration were unaltered by TRAM-34 treatment or genetic deletion stimulated Kcnn4 cells exhibited significantly decreased pCREB by guest on September 26, 2021 of KCa3.1 before (Supplemental Fig. 2B) and after stimulation compared with WT cells, as demonstrated by IF (Fig. 4A) and with RANKL, TNF, or a combination of both (Supplemental Western blot analysis (Fig. 4B). Furthermore, in qPCR studies we Fig. 2C). found that the expression of c-fos, a direct transcriptional target of 2/2 Collectively, both genetic deletion and pharmacological inhibition pCREB, was significantly reduced in Kcnn4 compared with of KCa3.1 significantly reduced the number of multinucleated cells WT cells during RANKL- and RANKL-TNF costimulation in the presence of TNF. (Fig. 4C). To evaluate the effects of reduced c-fos expression on the These data correlated with a reduction in mRNA expression of activity and protein expression of its transcriptional target Nfatc1, the osteoclast-specific genes Mmp9, Ctsk, and Acp5 in Kcnn42/2 we performed transcription factor binding assays in nuclear lysates and TRAM-34–treated cells compared with WT untreated controls of 36 h-stimulated BMMs. Results revealed a significant reduction 2/2 in the presence of RANKL in combination with TNF, as is evident of nuclear NFATc1 activity in RANKL-stimulated Kcnn4 from qPCR experiments (Fig. 2D). However, in the presence of compared with WT cells at this time point (Fig. 4D), whereas RANKL alone, only Ctsk and Acp5 were significantly reduced, NFATc1 activity in RANKL+TNF–stimulated BMMs was compa- 2/2 and no changes in Mmp9 expression were detected (Fig. 2C). Of rable in WT and Kcnn4 cells. To understand the differences in note, genetic and pharmacological inhibition of KCa3.1 were the dynamics of NFATc1 expression during RANKL-TNF costim- revealed to have similar effects on osteoclast-specific gene ex- ulation, we then performed Western blot studies using whole cell pression in the presence of RANKL, regardless of the presence of lysates at two different time points. Maximum NFATc1 expression TNF (Fig. 2C, 2D). in RANKL alone–stimulated cells was reached at 36 h and slightly decreased by 60 h incubation (Fig. 4E). When cells were incubated 2+ Inhibition of KCa3.1 reduces RANKL-induced Ca responses with RANKL and TNF, NFATc1 expression kept increasing by 60 h 2/2 To elucidate if KCa3.1 inhibition is specific to qualitative and stimulation in both, WT and Kcnn4 cells (Fig. 4F). In contrast quantitative changes in RANKL-induced Ca2+ responses, we next to activity, NFATc1 protein levels were significantly lower in 2/2 performed live cell Ca2+ imaging on cultured BMMs. In time- Kcnn4 cells compared with WT at both time points. In summary, lapse videos using confocal microscopy, we found that macro- these data show that deletion of Kcnn4 diminished pCREB during phages loaded with the Ca2+-sensitive dye Fluo-4 AM responded RANKL- and RANKL-TNF costimulation, followed by a significant with acute Ca2+ dynamics visualized by transient increases in decrease in c-fos mRNA and NFATc1 protein expression and activity. fluorescence intensity upon stimulation with RANKL (Fig. 3A, 3B). Inhibiting KCa3.1 function in these cultures via preincubation Discussion with TRAM-34 did not significantly reduce the relative number of In this study, we investigated the role of the K+ channel KCa3.1 responsive cells (Fig. 3C, left),or induce changes in the median during physiological and pathological osteoclast differentiation duration of Ca2+ transients (Fig. 3C, middle). However, we found under the influence of RANKL and the proinflammatory cytokine The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. RANKL-stimulated BMMs from Kcnn42/2 mice or treated with TRAM-34 in combination with TNF show decreased osteoclast formation correlated with decreased expression of osteoclast-specific genes. (A) Representative images of cytochemical TRAP staining of murine WT, Kcnn42/2, and WT+TRAM-34 (10 mM) BMMs cultured in indicated condition (scale bar, 100 mm). (B) Numbers of multinucleated TRAP+ cells (MNCs) (purple) ($3 nuclei) in these cultures (n = 4); (C) qPCR analysis showing normalized mRNA expression of osteoclast-specific genes Mmp9, Ctsk, Acp5 in RANKL; and (D) RANKL+TNF stimulated BMMs. One-way ANOVA followed by multiple comparison test was performed for all experiments (n = 4); shown are mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

TNF. We show that KCa3.1 plays a major role in osteoclast- ulation, (42, 43). In the current study, our microarray data reveal specific gene expression and formation of multinucleated cells that during RANKL stimulation no differences in Kir2.1 mRNA in both RANKL- and TNF-modulated osteoclastogenesis. (Kcnj2) expression between macrophages and osteoclasts are ap- It has previously been reported that TNF can induce an initial parent. Another study by Ohya et al. (34) suggested the presence increase in Kv1.3 (Kcna3) expression in murine macrophages, of a dominant-negative splice variant of Kcnn4 in mouse and peaking at 3 h poststimulation and decreasing afterward (43). Our human T cells, which forms a nonfunctional N-terminally trun- results are in agreement with this study, showing that Kv1.3 cated protein that can polymerize with the functional protein in the mRNA (Kcna3) levels are significantly lower than KCa3.1 cytosol, thereby interfering with membrane trafficking and for- (Kcnn4) at day 3 after stimulation. Of interest, the same study mation of functional channels. In the current study, we detected found the inwardly rectifying K+ channel Kir2.1, which we and both of these transcripts, the active (Kcnn4 a) as well as the others recently identified to be increased on anti-inflammatory dominant-negative variant (Kcnn4 b), to be expressed in macro- macrophages and microglia, is downregulated during TNF stim- phages and osteoclasts. This is to our knowledge the first time a 6 KCa3.1 REGULATES NFATc1 IN INFLAMMATORY OSTEOCLASTOGENESIS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Inhibition of KCa3.1 activity changes Ca2+ signaling in BMMs. (A) Representative snapshots obtained from time-lapse videos showing Ca2+ signals during acute stimulation with RANKL (100 ng/ml) in Fluo-4–loaded BMMs. (B) Representative fluorescence traces of Ca2+ signals during RANKL stimulation of a control (left) and a 3 mM TRAM-34–pretreated cell (right). (C) Analysis of Ca2+ responses in respective experiments. Unpaired Student t test and Mann–Whitney U test were performed when applicable. Overall response: control: n = 13, TRAM-34: n = 9; duration: control: n = 37, TRAM-34: n = 24; amplitude: control: n = 34, TRAM-34: n = 24. Shown are means 6 SEM. ****p , 0.0001. dominant-negative variant of Kcnn4 has been described in murine bers of multinucleated cells were reduced. The discrepancy in myeloid cells. Furthermore, in our IL-23 gene transfer model of osteoclast numbers between drug treatment and genetic deletion inflammatory arthritis, we found that KCa3.1 expression was upon RANKL stimulation that is abolished when TNF is present abundant on multinucleated cells on the bone surface (38). These may be indicative of compensation in the knockout model during findings extend the functional relevance of KCa3.1 expression on noninflammatory as opposed to inflammatory osteoclastogenesis. osteoclasts in inflammatory arthritis models in vivo. As demon- The addition of TNF, mimicking differentiation in inflammatory, strated in recent work by Kang et al. (32) using the collagen pathological conditions, seems to overrule this compensation, Ab-induced arthritis model, Kcnn42/2 mice exhibit decreased yielding a reduction in absolute numbers of multinucleated cells inflammation and lower disease scores compared with WT mice. ($3 nuclei) in Kcnn42/2 as well as TRAM-34–treated cells. The same study showed that KCa3.1 deletion contributes to de- Because TRAM-34 specificity for KCa3.1 is high, as previously creased formation of osteoclasts by diminishing cell fusion at a demonstrated, and proved to be concentration dependent, as certain stage after RANKL stimulation. In our study, Kcnn42/2 shown in our supplemental data (Supplemental Fig. 2), we rule out cells, despite yielding similar numbers of osteoclasts as defined by significant off-target effects of the drug (44, 45). Another aspect $3 nuclei, were smaller in size compared with WT cells, an ob- that seems to be puzzling at first is that we see no influence of servation that confirms a study by Kang et al. (32) demonstrating Kcnn4 deletion or TRAM-34 treatment on Mmp9 expression when that BMMs from Kcnn42/2 suffer from a defect in cell fusion cells were stimulated with RANKL. However, in inflammatory during RANKL-induced osteoclastogenesis. In the same setting, conditions using TNF in addition to RANKL, we do see a significant when pharmacologically inhibiting KCa3.1 function, total num- decrease of Mmp9 with both Kcnn4 deletion and TRAM-34. One The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Kcnn42/2 BMMs show decreased pCREB and expression of c-fos followed by reduced amplification and activity of NFATc1. (A) IF staining of pCREB (red) and DAPI (blue) of BMMs obtained from WT and Kcnn42/2 mice stimulated with RANKL or RANKL+TNF for 36 h visualized with confocal microscopy (n = 2, scale bar, 15 mm). (B) Western blot analysis of pCREB versus total CREB (tCREB) and b-actin; top: representative blot; bottom: densitometric analysis. One-way ANOVA with post hoc multiple comparison test was performed (n =2,p = 0.004). (C) qPCR analysis showing normalized c-fos mRNA expression of WT control and Kcnn42/2 BMMs cultivated in indicated conditions for 36 h. Student unpaired t test was performed (n = 4). (D) Transcription factor binding assays showing transcriptionally active NFATc1 protein obtained from nuclear lysates of 36 h stimulated WT and Kcnn42/2 BMMs in indicated conditions. One-way ANOVAwith post hoc multiple comparison test was performed (n =3,p = 0.0003). (E and F) Western blot analysis of NFATc1 protein expression obtained from whole cell lysates of WT and Kcnn42/2 BMMs stimulated for given time periods in indicated conditions; left: representative blots, right: densitometry analysis. One-way ANOVA with post hoc multiple comparison test was performed. (E) n =3, p = 0.0164; (F) n =3,p , 0.0001. Shown are mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. possible explanation could be that TNF in addition to RANKL ini- pathways. Another observation that supports the hypothesis that TNF tiates a signaling cascade that involves Mmp9 expression, which is to renders Ca2+ dynamics during osteoclastogenesis is that, interest- a higher degree dependent on Ca2+ and overwrites regular RANKL ingly, whereas the expression of NFATc1 in RANKL-stimulated 8 KCa3.1 REGULATES NFATc1 IN INFLAMMATORY OSTEOCLASTOGENESIS

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Supplemental figure 1: Kcnn4 is up-regulated during osteoclastogenesis. A) Hierarchical cluster analysis of RNA microarray data obtained from mouse BMMs before (control) and after treatment with RANKL (RANKL) displaying the differential gene expression patterns of a selected panel of K+ channels. B) Osteoclast formation was confirmed by cytochemical staining of TRAP (purple) in multinucleated giant cells (scale bar = 100µM) and C) expression of osteoclast-specific genes (OC genes) Ctsk and Mmp9 in arbitrary units (AU). D) Expression of K+ channels obtained from microarray raw data in arbitrary units (AU). E) qPCR analysis comparing KCa3.1 (Kcnn4 a) and Kv1.3 (Kcna3) mRNA expression in BMMs cultivated in respective conditions for three days; one-way ANOVA followed by post-hoc multiple comparison test was performed (N=3); *=P<0.05, **=P<0.01.

Supplemental figure 2: Concentration-response, cytotoxicity and viability studies for TRAM-34 and Kcnn4 knockout on macrophages and osteoclasts. A) Concentration-response curves for BMMs treated with indicated amounts of TRAM-34 prior to stimulation with RANKL or RANKL+TNF for three days; graph indicates formation of multinucleated cells (>3 nuclei); B) Cell viability/proliferation assays obtained from Alamar blue staining of BMMs isolated from WT and Kcnn4-/- mice, two, three and five days in culture medium; C) Alamar blue assays of WT control-treated, Kcnn4-/-, and BMMs pretreated with 10 µM TRAM-34 in the culture medium; shown are time points one day and three days after RANKL, TNF or RANKL+TNF stimulation (N=3), two-way ANOVA (P<0.0001 for both factors) with multiple comparison test was performed, *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001.

Supplemental figure 3: Inhibition of CaMKIV or CaN drastically decreases osteoclast formation and transcription of osteoclast-specific genes in the presence of TNF. A) Representative images of cytochemical TRAP staining of murine WT control BMMs (control), BMMs treated with 1 µM KN-93 (+KN-93) (to inhibit CaMKIV), or 100 nM cyclosporine A (+CsA) (to inhibit CaN) cultured in indicated conditions (N=3, scale bar = 100 µM). B) Numbers of multinucleated TRAP+ cells (purple) (>3 nuclei) obtained from A) (N=3); C) qPCR analysis showing normalized mRNA expression of osteoclast-specific genes (N=4); shown are means ± SEM, one-way ANOVA followed by multiple comparison test was performed, *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001. D) Western blot studies of BMMs stimulated with 100 ng/mL RANKL for indicated times showing phosphorylated (pCaMKIV) and total CaMKIV (tCaMKIV) versus β-actin; left: control stimulated, middle: pre-treated with 3 µM TRAM-34, right: in culture medium with increased K+ concentration; blots are representative for two independent experiments.