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FEBS Letters 579 (2005) 832–838 FEBS 29223

Phosphodiesterase inhibitors stimulate osteoclast formation via TRANCE/RANKL expression in osteoblasts: possible involvement of ERK and p38 MAPK pathways

Masamichi Takamia,1, Eun Sook Chob,1, Soo Young Leec, Ryutaro Kamijoa, Mijung Yimb,* a Department of Biochemistry, School of Dentistry, Showa University, Tokyo 142-8555, Japan b College of Pharmacy, Sookmyung WomenÕs University, Seoul 140-742, Republic of Korea c Division of Molecular Life Sciences and Center for Cell Signaling Research, Ewha WomenÕs University, Seoul 120-750, Republic of Korea

Received 20 October 2004; revised 1 December 2004; accepted 14 December 2004

Available online 12 January 2005

Edited by Lukas Huber

several factors, such as 1,25-dihydroxyvitamin D Abstract Phosphodiesterases (PDEs) are that degrade 3 intracellular cAMP. In the present study, 3-isobutyl-1-methyl- [1,25(OH)2D3], parathyroid hormone (PTH), interleukin (IBMX) and , PDE inhibitors, induced (IL)-6 plus soluble IL-6 , prostaglandin E2 (PGE2), osteoclast formation in cocultures of mouse bone marrow cells and [2–4]. Those factors induce the expression of and calvarial osteoblasts. These inhibitors induced the expression TNF-related activation-induced cytokine (TRANCE, also of the osteoclast differentiation factor, TNF-related activation known as RANKL, ODF, or OPGL) in osteoblasts, which induced cytokine (TRANCE, identical to RANKL, ODF, and triggers osteoclast differentiation [5–8]. Osteoblasts also pro- OPGL), in calvarial osteoblasts. IBMX induced phosphorylation duce osteoprotegerin (OPG), a decoy receptor for TRANCE, of extracellular signal-regulated kinase (ERK) and p38 mitogen- to inhibit osteoclast formation [9]. activated protein kinase (MAPK) in osteoblasts. Induction of Cyclic monophosphate (cAMP) is a secondary TRANCE expression by IBMX was partially suppressed by messenger in intracellular signaling cascades and elevation of the inhibitors of (PKA), ERK, and p38 MAPK, suggesting that activation of ERK and p38 MAPK, as well as intracellular cAMP levels activates protein kinase A (PKA) PKA, is involved in TRANCE expression by IBMX. Osteoblasts and controls gene expression and cell function [10,11]. This expressed PDE4, a PDE subtype, and , a selective pathway is known to induce TRANCE expression in osteo- inhibitor of PDE4, induced TRANCE expression. These results blasts following stimulation with PTH and PGE2 [12–14]. suggest that PDE4 is a key regulator of TRANCE expression in Intracellular cAMP is generated by adenylate cyclase from osteoblasts, which in turn controls osteoclast formation. (ATP) as a substrate, whereas 2005 Federation of European Biochemical Societies. Published cAMP-specific phosphodiesterases (PDEs) degrade cAMP by by Elsevier B.V. All rights reserved. hydrolysis [10,11]. Thus, intracellular cAMP gradients are gov- erned by a balance between its generation by adenylate cyclase Keywords: Osteoblast; Osteoclast; Cyclic adenosine and degradation by PDEs. monophosphate; Phosphodiesterase; TNF-related activation- induced cytokine The PDE family consists of 11 isozymes, PDE1 to 11, and those involved in the degradation of cAMP are PDE1, 2, 3, 4, 7, 8, 10, and 11, with some PDE isozymes further classified into subtypes [11]. Various low molecular weight inhibitors of PDEs have been used as tools to study their biological roles in bone metabolism [15–17]. Pentoxifylline and 3-isobutyl-1- 1. Introduction methylxanthine (IBMX) are PDE inhibitors that inhibit all members of the PDE family, and induce an elevation of intra- Osteoclasts are multinucleated giant cells responsible for cellular cAMP levels by inhibiting cAMP hydrolysis [11]. It has bone resorption [1]. Osteoblasts, as well as stromal cells, are been reported that pentoxifylline and rolipram, a selective essentially required for osteoclastogenesis through cell–cell PDE4 inhibitor, increase systemic bone-mass in mice [15]. interactions with osteoclast precursors of monocyte/macro- Other PDE4 inhibitors have also been reported to show a ther- phage lineage [2,3]. In cocultures of mouse bone marrow cells apeutic effect against bone loss in some animal osteopenia and calvarial osteoblasts, osteoclasts are formed in response to models [16,17]. Therefore, PDE inhibitors are considered to be promising candidates for anti-osteoporosis drug therapies, however, their effects on osteoclastogenesis have not been fully * Corresponding author. Fax: +82 2 710 9871. elucidated. E-mail address: [email protected] (M. Yim). In the present study, the effects of PDE inhibitors on osteo- 1 M. Takami and E. S. Cho contributed equally to this work. clast formation were examined using a mouse coculture system. We found that IBMX, pentoxifylline, and the PDE4- Abbreviations: PDE, phosphodiesterase; TRANCE, TNF-related acti- selective inhibitor, rolipram, strongly induced osteoclast vation-induced cytokine; OPG, osteoprotegerin; PTH, parathyroid hormone; IBMX, 3-isobutyl-1-methylxanthine; PKA, protein kinase formation in the cocultures. Further, TRANCE mRNA and A; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein expression levels were elevated by IBMX and rolipram protein kinase in calvarial osteoblasts. Our results also suggested that

0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2004.12.066 M. Takami et al. / FEBS Letters 579 (2005) 832–838 833

TRANCE expression by IBMX and rolipram is mediated not ence or absence of various concentrations of PDE inhibitors or only by PKA, but also by extracellular signal-regulated kinase 1,25(OH)2D3 (10 nM) in 96-well culture plates (CORNING, MA, (ERK) and p38 mitogen-activated protein kinase (MAPK). USA). After 6 days of culture, cells were fixed and stained for tar- trate-resistant acid phosphatase (TRAP), a marker of osteo- Thus, PDE4 is considered to act as a key regulator of clasts, as described previously [18]. TRANCE expression in osteoblasts. 2.3. Pit formation assay Mouse bone marrow cells (1 · 105 cells) and calvarial osteoblasts 3 2. Materials and methods (5 · 10 cells) were cocultured in the presence or absence of IBMX (50 lM) on dentine slices (0.3 mm thick, 4 mm in diameter) placed in 48-well culture plates for 6 days, as described previously [18]. The slices 2.1. Reagents were then recovered and stained with toluidine blue to visualize resorp- IBMX and toluidine blue were purchased from Wako Pure Chem- tion pits. ical Industries Ltd. (Tokyo, Japan). PTH (human, 1-34) was pur- chased from PEPTIDE Institute, Inc. (Osaka, Japan). PD98059 and SB203580 were obtained from Calbiochem (San Diego, CA, 2.4. Northern-blot analysis USA). Antibodies against ERK, phospho-ERK, p38 and phospho- Calvarial osteoblasts in 60-mm diameter dishes were cultured with p38 were purchased from Cell Signaling Technology (Beverly, agents for the indicated periods and then subjected to total RNA MA, USA). All other reagents were from Sigma–Aldrich (St. Louis, extraction using Trizol reagent (Invitrogen, CA, USA) according to MO, USA). the manufacturerÕs protocols. Total RNA (20 lg) was electrophoresed on 1.2% agarose–formaldehyde gels, transferred to nylon membrane filters (Hybond N+, Amersham Biosciences, Buckinghamshire, UK), 2.2. Cells and culture system and hybridized with 32P-labeled cDNA probes. After the final wash, Primary calvarial osteoblasts were obtained from the calvariae of the membranes were exposed to X-ray film (BioMax, Kodak, NY, neonatal ddY mice (Japan SLC Inc., Shizuoka, Japan) using 0.1% col- USA) at 70 C. lagenase and 0.2% dispase [18]. Bone marrow cells were obtained from the long bones of 4- to 6-week-old ddY male mice. UAMS-32 cells were kindly provided by Dr. Charles A. OÕBrien (University of Arkan- 2.5. Flow cytometry analysis sas for Medical Sciences, USA). To examine osteoclast formation, Osteoblasts treated with or without IBMX (50 lM) for 24 h were mouse bone marrow cells (1 · 105 cells) were cocultured with calvarial stained with Fc-conjugated soluble TRANCE receptor (RANK) osteoblasts (5 · 103 cells), or UAMS-32 cells (5 · 103 cells) in the pres- (kindly provided by Prof. Yongwon Choi, University of Pennsylvania,

Fig. 1. Effects of PDE inhibitors on osteoclast formation. (A and B) Dose-dependent effects of IBMX and pentoxifylline on osteoclast formation in cocultures. Mouse bone marrow cells and calvarial osteoblasts were cocultured in the presence of the indicated concentrations of IBMX (A) or pentoxifylline (B) for 6 days. Cells were then fixed and stained for TRAP. TRAP-positive (+) multinucleated cells (MNCs) were counted. Data are expressed as means + S.D. of triplicate cultures. (C) Photographs of cocultures (upper panels) and dentin slices (lower panels), on which cells were cocultured with (+) or without ()50lM of IBMX for 6 days. Cells were fixed and stained for TRAP, which appear as dark-stained cells. Resorption pits were stained with toluidine blue after removing the cells and appear as dark dots. (D) Expression of calcitonin receptor (CTR) and cathepsin K mRNA in cocultured cells. Bone marrow cells and calvarial osteoblasts were cocultured for 6 days with (+) or without ()50lM of IBMX. Total RNA was then isolated from the cells and cDNA templates prepared with (+) or without () reverse transcriptase (RT). mRNA expression was determined by RT-PCR using specific primers designed for each gene. Bar = 200 lm. 834 M. Takami et al. / FEBS Letters 579 (2005) 832–838

USA) followed by phycoerythrin-conjugated anti-IgG antibody (BD Pharmingen, San Diego, CA, USA). The stained cells were analyzed by flow cytometry.

2.6. RT-PCR analysis Total RNA (1 lg) was reverse-transcribed using Superscript II (Invitrogen, CA, USA) according to the manufacturerÕs protocols. Ali- quots of the obtained cDNA pool were subjected to PCR amplifica- tion with Go Taq DNA polymerase (Promega Co., WI, USA). Primers for mouse PDE4s, TRANCE, and GAPDH used in this study are as follows: PDE4A, 50-ggaactcacacacctgtcg-30 (forward), 50- gttcttgtgctaagaggtcc-30(reverse); PDE4B, 50-tggaaatcctggctgccat-30 (forward), 50-tccacagaagctgtgtgct-30 (reverse); PDE4C, 50-tggtatcagag- taggattcc-30 (forward), 50-ctctgtgtaaaccttggctg-30 (reverse); PDE4D, 50-cggaactcgctctgatgt-30 (forward), 50-acagaggcgttgtgcttg-30 (reverse); TRANCE, 50-ccagccatttgcacacctc-30 (forward), 50-agcagggaagggttgg- aca-30 (reverse); calcitonin receptor (CTR), 50-tttcaagaaccttagctgcca- gag-30 (forward), 50-caaggcacggacaatgttgagaag-30 (reverse); cathepsin K, 50-cttccaatacgtgcagcaga-30 (forward), 50-acgcaccaatatcttgcacc-30 (reverse); and GAPDH, 50-gaaggtcggtgtgaacggatttggc-30 (forward), 50-catgtaggccatgaggtccaccac-30 (reverse). The PCR program was as follows: 32 (all mouse PDE4s, TRANCE, CTR, and cathepsin K) or 28 (GAPDH) cycles, after an initial denaturation step at 94 C for 3 min, then denaturation at 94 C for 30 s, annealing at 58 C for 45 s, and extension at 72 C for 60 s, with a final extension at 72 C for 10 min.

2.7. Immunoblot analysis Total cell lysates were isolated, separated by SDS–PAGE, and trans- ferred onto Immobilon-P membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat-milk in TBS-T (150 mM NaCl, 20 mM Tris, pH 7.4, and 0.1% Tween 20), and then immunostained with anti-phospho p38 (1:1000), anti-phospho ERK (1:1000), anti-p38 (1:1000) or with anti-ERK antibody (1:1000) fol- lowed by secondary horseradish peroxidase-conjugated antibody (1:5000). The membranes were developed using an enhanced chemilu- minescence detection kit (Amersham Biosciences).

3. Results

3.1. IBMX stimulates osteoclast formation in mouse coculture system Fig. 2. Requirement of TRANCE for osteoclast formation induced by We first examined the effects of the PDE inhibitors, IBMX IBMX. (A) Mouse bone marrow cells and calvarial osteoblasts were and pentoxifylline, on osteoclast formation using cocultures cocultured with (+) or without ()50lM of IBMX in the presence (+) or absence () of OPG (100 lg/ml) for 6 days. Cells were then fixed of mouse bone marrow cells and calvarial osteoblasts. Each and stained for TRAP. Data are expressed as means + S.D. of inhibitor induced TRAP-positive multinucleated cell (MNC) triplicate cultures. (B) Calvarial osteoblasts were treated with indicated formation in a dose-dependent manner (Fig. 1A–C). The concentrations of IBMX for 3 h. Total RNA was then extracted from mRNA expression levels of CTR and cathepsin K, osteoclast the osteoblasts and the expression of TRANCE mRNA was examined markers, were increased in the cultures treated with IBMX by Northern-blot analysis. (C) Osteoblasts were treated with (+) or without () IBMX (50 lM) for 24 h. Then, cells were harvested and (Fig. 1D). When the cells were cocultured in the presence of stained with Fc-conjugated soluble TRANCE receptor (RANK) which IBMX (50 lM) on dentine slices, resorption pits were formed binds to TRANCE followed by phycoerythrin-conjugated anti-IgG (Fig. 1C). These results indicate that both IBMX and pentox- antibody. The stained cells were analyzed by flow cytometry. ifylline strongly induce osteoclasts in a mouse coculture sys- tem.

3.2. Induction of TRANCE expression in calvarial osteoblasts is not shown). IBMX dose-dependently stimulated TRANCE involved in osteoclast formation mRNA expression in calvarial osteoblasts, with the maxi- Osteoclast formation induced by IBMX was completely mum effect seen at 100 lM(Fig. 2B). In agreement with inhibited by addition of OPG, a decoy receptor for this, analysis of osteoblastic cell surface by flow cytometry TRANCE (Fig. 2A). Furthermore, addition of soluble revealed that TRANCE expression is upregulated in protein TRANCE receptor (RANK) also inhibited osteoclast forma- level after IBMX treatment (Fig. 2C). tion induced by IBMX (data not shown). These data suggest that TRANCE is involved in the osteoclast formation in- 3.3. Inhibitors of PKA, ERK and p38 MAPK suppress duced by IBMX. To determine whether IBMX induces TRANCE expression induced by IBMX TRANCE expression, calvarial osteoblasts were treated with To identify the signaling pathways used by IBMX to in- various concentrations of IBMX for 3 h. IBMX elevated duce TRANCE expression, the roles of the PKA, ERK, intracellular cAMP levels in osteoblasts within 3 h (data and p38 MAPK signaling cascades in TRANCE expression M. Takami et al. / FEBS Letters 579 (2005) 832–838 835

Fig. 3. Effects of H89, PD98059, and SB203580 (inhibitors of PKA, ERK, and p38 MAPK, respectively) on TRANCE mRNA expression (A, C) and intracellular signal transduction (B) in osteoblasts. (A and C) Calvarial osteoblasts were preincubated in the absence or presence of 30 lM of H89, 50 lM of PD98059, or 50 lM of SB203580 for 30 min, and then treated with (+) or without ()50lM of IBMX (A) or 100 nM PTH (C) for 3 h. Total RNA was extracted from the cells and the expressions of TRANCE and GAPDH mRNA were examined by Northern-blot analysis. (B) Osteoblasts were treated with IBMX (50 lM) for indicated times. Then, cell lysates were harvested, separated by SDS–PAGE, and transferred onto PVDF membranes. Phospho-ERK, ERK, phospho-p38 MAPK, and p38 MAPK were detected by immunoblotting using specific antibodies. were examined. Consistent with previous reports [12–14], 3.4. PDE4 is a key regulator of TRANCE expression in pretreatment of calvarial osteoblasts with a PKA inhibitor, calvarial osteoblasts H89, decreased TRANCE mRNA expression levels induced To determine the PDE isozymes responsible for TRANCE by IBMX (Fig. 3A). Interestingly, a MAPK/ERK kinase expression in calvarial osteoblasts, effects of various isozyme- (MEK) inhibitor, PD98059, and a p38 MAPK inhibitor, selective PDE inhibitors on osteoclast formation and SB203580, also decreased TRANCE mRNA expression lev- TRANCE mRNA expression in calvarial osteoblasts were els induced by IBMX (Fig. 3A). PD98059 and SB203580, examined. A PDE4 selective inhibitor, rolipram, dose-depen- but not H89 alone slightly decreased endogenous TRANCE dently induced osteoclast formation and TRANCE mRNA mRNA expression levels (Fig. 3A). Immunoblot analysis expression in calvarial osteoblasts (Fig. 4A–C). , using specific antibodies against phospho-ERK and phos- EHNA, , , and (selective pho-p38 MAPK indicated that IBMX induced the activation inhibitors of PDE1, PDE2, PDE3, PDE5 and 8, PDE5 and of ERK and p38 MAPK pathways in osteoblasts (Fig. 3B). 9, respectively) did not induce osteoclast formation or These results suggest that IBMX regulates TRANCE expres- TRANCE mRNA expression in calvarial osteoblasts as sion in osteoblasts via activation of the ERK and p38 strongly as IBMX and rolipram (Fig. 4A and C). Similar to MAPK signaling pathways, as well as PKA. the results by IBMX, H89, PD98059, and SB203580 decreased Since PTH is known to stimulate TRANCE mRNA the TRANCE mRNA expression levels induced by rolipram expression via cAMP accumulation [12,14], we examined (Fig. 4D). These results suggest that TRANCE regulation in the effects of PD98059 and SB203580 on TRANCE mRNA calvarial osteoblasts is attributable to PDE4. expression induced by PTH. These inhibitors also decreased the expression levels of TRANCE mRNA induced by PTH 3.5. PDEs regulate TRANCE expression in cells of osteoblastic (Fig. 3C), suggesting that ERK and p38 MAPK signaling lineage pathways are involved in PTH-induced TRANCE expres- Based on the results shown above, we compared the expres- sion. sion patterns of PDE4 subtypes in calvarial osteoblasts and the 836 M. Takami et al. / FEBS Letters 579 (2005) 832–838 mouse bone-marrow stromal cell line, UAMS-32. Calvarial 4. Discussion osteoblasts, as well as UAMS-32 cells, expressed PDE4A, B, and 4D, however, not 4C (Fig. 5A). Both calvarial osteoblasts In the present study, we demonstrated that PDE inhibitors and UAMS-32 cells supported osteoclast formation, and in- induce osteoclast formation by inducing TRANCE expression duced TRANCE mRNA expression in response to IBMX in calvarial osteoblasts and UAMS-32 cells, suggesting that and 1,25(OH)2D3, an activator of vitamin D receptor-medi- PDEs are the key molecules that negatively regulate TRANCE ated pathway independently of PKA and MAPK (Fig. 5B expression by degrading cAMP. Our results also suggested that and C). These results suggest that not only vitamin D receptor PDE4 mainly regulates cAMP levels and TRANCE expression but also PDEs regulate TRANCE expression in cells of osteo- in osteoblasts. blastic lineage. In contrast to our results, previous reports have shown the anabolic effects of PDE inhibitors in vivo [15–17]. Kinoshita et al. [15] have reported that pentoxifylline and rolipram in- creased bone mass in animals that were given those drugs in daily injections. Interestingly, PTH as well as PDE inhibitors induce osteoclast formation in a mouse coculture system. However, injections of PTH to animals once daily produce a net anabolic effect in vivo [14,19]. Furthermore, continu- ous exposure to PTH leads to a coupled increase in bone formation and bone resorption, with a net loss of bone mass [19]. Therefore, PDE inhibitors may induce bone loss if the animals are administrated continuously. Additional analysis of PDE inhibitor-administrated animals under various con- ditions is necessary to understand the effects of PDEs on bone metabolism. Elevated intracellular cAMP mainly activates PKA and this pathway has been implicated in TRANCE-mediated osteoclast formation [12–14]. In agreement with this, our experiments showed that the PKA inhibitor reduced the TRANCE mRNA expression induced by IBMX. Recently, Rawadi et al. [20] reported that ERK1/2 and p38 MAPK are activated by pentoxifylline to promote the differentiation of osteoblasts. Indeed, our results showed that IBMX acti- vated ERK and p38 MAPK pathways, and the ERK inhib- itor, PD98059, and p38 MAPK inhibitor, SB203580, reduced TRANCE mRNA expression induced by IBMX and roli- pram. Furthermore, these MAPK inhibitors partially sup- pressed PTH-induced elevation of TRANCE expression as well. These results suggest that an alternative signaling path- way contributes to TRANCE expression via ERK and p38 MAPK activation following the cAMP elevation in osteo- blasts. Further study will elucidate the precise relationships between PKA and MAPK. In the present coculture system, only the PDE4 inhibitor rolipram, and not other inhibitors selective for PDE isozymes, b Fig. 4. Effects of PDE isozyme selective inhibitors on osteoclast formation and TRANCE mRNA expression in calvarial osteoblasts. (A and B) Mouse bone marrow cells and calvarial osteoblasts were cocultured in the absence or presence of vinpocetine (30 lM), EHNA (10 lM), milrinone (10 lM), rolipram (10 lM), dipyridamole (30 lM), zaprinast (100 lM) (selective inhibitors for PDE1, 2, 3, 4, 5 and 8, 5 and 9, respectively), pentoxifylline (200 lM), or IBMX (50 lM) (non- selective inhibitors) for 6 days (A). Mouse bone marrow cells and calvarial osteoblasts were cocultured with indicated concentrations of rolipram for 6 days (B). Cells were then fixed and stained for TRAP. Data are expressed as means + S.D. of triplicate cultures. (C) Calvarial osteoblasts were treated with vinpocetine, EHNA, milrinone, rolipram, dipyridamole, zaprinast, or IBMX for 3 h. Expressions of TRANCE and GAPDH mRNA were determined by RT-PCR. (D) Calvarial osteoblasts were preincubated in the absence () or presence (+) of 30 lM of H89, 50 lM of PD98059, or 50 lM of SB203580 for 30 min, and then treated with (+) or without ()10lM of rolipram for 3 h. Total RNA was extracted from the cells and the expressions of TRANCE and GAPDH mRNA were examined by Northern-blot analysis. M. Takami et al. / FEBS Letters 579 (2005) 832–838 837

animals given PDE inhibitors will contribute to the develop- ment of anti-osteoporosis drugs based on PDE activities.

Acknowledgments: We thank Dr. Charles A. OÕBrien (University of Arkansas for Medical Sciences, USA) for providing the UAMS-32 cells. We also thank Dr. Yongwon Choi (University of Pennsylvania, USA), Dr. Nacksung Kim (Chonnam National University, Korea), Dr. Dong-Seok Lee (Korea Research Institute of Bioscience and Biotechnology, Korea), and Dr. Kunhong Kim (Yonsei University, Korea) for technical support.

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