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Essential Role of p38 Mitogen-activated Protein Kinase in Cathepsin K Gene Expression during Osteoclastogenesis through Association of NFATc1 and PU.1

2004; Elsevier BV; Volume: 279; Issue: 44 Linguagem: Inglês

10.1074/jbc.m408795200

ISSN

1083-351X

Autores

Masahito Matsumoto, Masakazu Kogawa, Seiki Wada, Hiroshi Takayanagi, Masafumi Tsujimoto, Shigehiro Katayama, Koji Hisatake, Yasuhisa Nogi,

Tópico(s)

Bone health and treatments

Resumo

The receptor activator of NF-κB ligand (RANKL) induces various osteoclast-specific marker genes during osteoclast differentiation mediated by mitogen-activated protein (MAP) kinase cascades. However, the results of transcriptional programming of an osteoclast-specific cathepsin K gene are inconclusive. Here we report the regulatory mechanisms of RANKL-induced cathepsin K gene expression during osteoclastogenesis in a p38 MAP kinase-dependent manner. The reporter gene analysis with sequential 5′-deletion constructs of the cathepsin K gene promoter indicates that limited sets of the transcription factors such as NFATc1, PU.1, and microphthalmia transcription factor indeed enhance synergistically the gene expression when overexpressed in RAW264 cells. In addition, the activation of p38 MAP kinase is required for the maximum enhancement of the gene expression. RANKL-induced NFATc1 forms a complex with PU.1 in nuclei of osteoclasts following the nuclear accumulation of NFATc1 phosphorylated by the activated p38 MAP kinase. These results suggest that the RANKL-induced cathepsin K gene expression is cooperatively regulated by the combination of the transcription factors and p38 MAP kinase in a gradual manner. The receptor activator of NF-κB ligand (RANKL) induces various osteoclast-specific marker genes during osteoclast differentiation mediated by mitogen-activated protein (MAP) kinase cascades. However, the results of transcriptional programming of an osteoclast-specific cathepsin K gene are inconclusive. Here we report the regulatory mechanisms of RANKL-induced cathepsin K gene expression during osteoclastogenesis in a p38 MAP kinase-dependent manner. The reporter gene analysis with sequential 5′-deletion constructs of the cathepsin K gene promoter indicates that limited sets of the transcription factors such as NFATc1, PU.1, and microphthalmia transcription factor indeed enhance synergistically the gene expression when overexpressed in RAW264 cells. In addition, the activation of p38 MAP kinase is required for the maximum enhancement of the gene expression. RANKL-induced NFATc1 forms a complex with PU.1 in nuclei of osteoclasts following the nuclear accumulation of NFATc1 phosphorylated by the activated p38 MAP kinase. These results suggest that the RANKL-induced cathepsin K gene expression is cooperatively regulated by the combination of the transcription factors and p38 MAP kinase in a gradual manner. Osteoclasts that play pivotal roles in bone morphogenesis, remodeling and resorption, differentiate from the hematopoietic myeloid precursors of macrophage/monocyte lineage (1Suda T. Takahashi N. Udagawa N. Jimi E. Gillespie M.T. 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Steingrimsson E. Copeland N.G. Jenkins N.A. Arnheiter H. Cell. 1993; 74: 395-404Abstract Full Text PDF PubMed Scopus (950) Google Scholar) expressed in osteoclast precursors at the downstream of the RANKL signaling. The interaction of MITF with either PU.1 or PU.1-interacting protein allows efficient induction of an osteoclast-specific marker of the tartrate-resistant acid phosphatase (TRAP) gene in a synergistic manner (22So H. Rho J. Jeong D. Park R. Fisher D.E. Ostrowski M.C. Choi Y. Kim N. J. Biol. Chem. 2003; 278: 24209-24216Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 23Matsumoto M. Sudo T. Saito T. Osada H. Tsujimoto M. J. Biol. Chem. 2000; 275: 31155-31161Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). In mutant mice models, these transcription factors are disrupted or mutated but are of prime importance in showing their essential roles for osteoclast differentiation or its function, and these mice exhibit osteopetrosis because of defects in multinucleated osteoclasts (17Wang Z.Q. Ovitt C. Grigoriadis A.E. Mohle-Steinlein U. Ruther U. Wagner E.F. Nature. 1992; 360: 741-745Crossref PubMed Scopus (810) Google Scholar). Recently, it has been revealed that the nuclear factor of activated T cells (NFAT) c1 transcription factor was induced by RANKL stimulation and then activated the TRAP gene promoter synergistically with c-Fos (24Ishida N. Hayashi K. Hoshijima M. Ogawa T. Koga S. Miyatake Y. Kumegawa M. Kimura T. Takeya T. J. Biol. Chem. 2002; 277: 41147-41156Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 25Takayanagi H. Kim S. Koga T. Nishina H. Isshiki M. Yoshida H. Saiura A. Isobe M. Yokochi T. Inoue J. Wagner E.F. Mak T.W. Kodama T. Taniguchi T. Dev. Cell. 2002; 3: 889-901Abstract Full Text Full Text PDF PubMed Scopus (2003) Google Scholar). Thus, the inducibility of the osteoclast-specific genes is controlled by the combination of the RANKL-induced and noninducible transcription factors in a gradual manner. A cysteine protease of the osteoclast-specific marker cathepsin K, which is capable of hydrolyzing extracellular bone matrix proteins, is highly expressed in osteoclasts (26Saftig P. Hunziker E. Wehmeyer O. Jones S. Boyde A. Rommerskirch W. Moritz J.D. Schu P. von Figura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13453-13458Crossref PubMed Scopus (759) Google Scholar). Pycnodysostosis is a human disease caused by congenital cathepsin K deficiency and indicates a crucial role of cathepsin K in the functional maturation of osteoclasts (27Gelb B.D. Shi G.P. Chapman H.A. Desnick R.J. Science. 1996; 273: 1236-1238Crossref PubMed Scopus (859) Google Scholar). The expression of the cathepsin K gene is controlled physiologically in vivo and in vitro by MITF (28Motyckova G. Weilbaecher K.N. Horstmann M. Rieman D.J. Fisher D.Z. Fisher D.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5798-5803Crossref PubMed Scopus (194) Google Scholar). The mutant mi/mi mice that express a dominant negative form of MITF have the following characteristics, unpigmented small eyes, several intrinsic defects in melanocytes, mast cells, and multinucleated osteoclasts leading to osteopetrosis (21Hodgkinson C.A. Moore K.J. Nakayama A. Steingrimsson E. Copeland N.G. Jenkins N.A. Arnheiter H. Cell. 1993; 74: 395-404Abstract Full Text PDF PubMed Scopus (950) Google Scholar), which indicate essential roles of MITF in development of several cell type lineages. Cathepsin K mRNA is still expressed in osteoclasts derived from the mutant mice (28Motyckova G. Weilbaecher K.N. Horstmann M. Rieman D.J. Fisher D.Z. Fisher D.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5798-5803Crossref PubMed Scopus (194) Google Scholar, 29Luchin A. Purdom G. Murphy K. Clark M.Y. Angel N. Cassady A.I. Hume D.A. Ostrowski M.C. J. Bone Miner. Res. 2000; 15: 451-460Crossref PubMed Scopus (109) Google Scholar), suggesting a possible involvement of at least another modulator(s) upon the gene expression. In osteoclast differentiation, the RANKL signaling is mainly mediated by recruiting adaptor molecules, TRAF proteins, that activate the mitogen-activated protein (MAP) kinase cascades (14Teitelbaum S.L. Science. 2000; 289: 1504-1508Crossref PubMed Scopus (3104) Google Scholar, 30Wong B.R. Josien R. Lee S.Y. Vologodskaia M. Steinman R.M. Choi Y. J. Biol. Chem. 1998; 273: 28355-28359Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). Three distinct MAPs kinases, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase, and p38 MAP kinase, identified in mammalian cells, are implicated as mediators to transmit nuclear signaling for cell growth and differentiation in response to stress and various cytokines (31Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1663) Google Scholar, 32Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (691) Google Scholar). Each of the kinases can activate specific transcription factors through direct phosphorylation prior to efficient expression of target genes. The p38 MAP kinase family members, consisting of p38α, p38β (33Jiang Y. Chen C. Li Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. Chem. 1996; 271: 17920-17926Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar), p38γ (34Li Z. Jiang Y. Ulevitch R.J. Han J. Biochem. Biophys. Res. Commun. 1996; 228: 334-340Crossref PubMed Scopus (353) Google Scholar), and p38δ (35Jiang Y. Gram H. Zhao M. New L. Gu J. Feng L. Di Padova F. Ulevitch R.J. Han J. J. Biol. Chem. 1997; 272: 30122-30128Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar) identified so far, serve as key determinants in cytokine-induced cell differentiation. In fact, we and others (23Matsumoto M. Sudo T. Saito T. Osada H. Tsujimoto M. J. Biol. Chem. 2000; 275: 31155-31161Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 36Li X. Udagawa N. Itoh K. Suda K. Murase Y. Nishihara T. Suda T. Takahashi N. Endocrinology. 2002; 143: 3105-3113Crossref PubMed Scopus (171) Google Scholar) have reported that p38α MAP kinase activated by MAP kinase kinase 3 (MKK3) or MKK6, downstream of the RANKL signaling pathway, is critical for the relatively early phase of osteoclast differentiation. Recently, Mansky et al. (37Mansky K.C. Sankar U. Han J. Ostrowski M.C. J. Biol. Chem. 2002; 277: 11077-11083Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) have reported that p38α MAP kinase phosphorylates MITF, resulting in increasing a synergistic expression level of the TRAP gene. These findings suggest that the critical roles of p38 MAP kinase are to trigger and enhance the coordinated induction of the osteoclast-specific gene(s) through the modification of the limited sets of the transcription factor(s). However, the molecular mechanism of the cathepsin K gene expression remains to be fully defined in the context of RANKL-induced osteoclast differentiation mediated by the MAP kinase cascades. This study shows that treatment with SB203580, a specific inhibitor of the p38 MAP kinase, markedly suppresses not only RANKL-induced cathepsin K gene expression in osteoclasts but osteoclast differentiation. After the analyses of the inducible mechanism of the RANKL-induced cathepsin K gene, we found here for the first time that NFATc1 interacts directly with PU.1, and these factors activate the gene promoter in concert with MITF. p38 MAP kinase phosphorylates NFATc1 but not PU.1 and thereby invokes enhancement of nuclear accumulation of NFATc1 and of the transcriptional activation of the cathepsin K gene promoter. These findings suggest that the transcriptional program of the RANKL-induced osteoclast-specific genes is controlled by limited sets of transcription factors at distinct stages of osteoclast differentiation. These studies give more detailed molecular analyses of the cathepsin K gene transcription leading the cells to undergo terminal differentiation of osteoclasts. Materials—Human recombinant soluble RANKL (sRANKL) was purchased from PeproTech (London, UK). Recombinant human macrophage colony-stimulating factor (M-CSF) was kindly provided by Morinaga Milk Industry Co., Ltd. (Tokyo, Japan). Polyclonal antibodies against p38 MAP kinase and phosphorylated p38 MAP kinase (Thr180/Tyr182) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Polyclonal antibody against PU.1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-NFATc1 monoclonal antibody was purchased from BD Biosciences. Alexa Fluor 546 goat antimouse and 488 goat anti-rabbit IgG antibodies were purchased from Molecular Probes, Inc. (Eugene, OR). Reverse transcription-PCR kit was obtained from Invitrogen. SB203580 and PD98059 were purchased from Calbiochem. Cell Culture—Bone marrow cells were prepared by removing femurs and tibias from 6- to 8-week-old C5B/B6N mice and flashing the bone marrow cavity with α-modified minimum Eagle's medium (Sigma) containing 10% fetal bovine serum (FBS; Biological Industries, Israel) and 50 μg/ml penicillin/streptomycin (ICN Biomedical, Inc. OH). After lysing the erythrocytes in lysing buffer (17 mm Tris, pH 7.65, 0.75% NH4Cl), cells were seeded at 1.5 ×106 cells/well (0.5 ml) in 24-well plates in the presence of M-CSF. After a 3-day incubation, nonadherent cells were removed from the culture by pipetting and washed with phosphate-buffered saline (PBS). Adherent cells were further incubated in the presence of M-CSF and sRANKL for 5–6 days. The culture medium was replaced every 3 days with fresh complete medium. The cells were then washed and subjected to immunostaining, reverse transcription-PCR (RT-PCR), and immunoblot analysis. The murine RAW264 cells (RIKEN, RCB0535) were maintained in α-modified minimum essential medium supplemented with 10% FBS (JRH Biosciences, Lenexa, KS) and 50 μg/ml penicillin/streptomycin as described previously. The HEK293 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS and 50 μg/ml penicillin/streptomycin. Peripheral blood mononuclear cells (PBMC) were prepared from human heparinized blood by the Ficoll-Paque gradient centrifugation method and were incubated with human M-CSF. After 7 days, adherent macrophages were cultured with sRANKL for 3–5 days, and human osteoclasts were subjected to chromatin immunoprecipitation (ChIP) assay. Detection of Cathepsin K mRNA Expression by RT-PCR—Total RNA for cDNA synthesis was isolated from murine bone marrow cells and cultured cells as described previously (38Matsumoto M. Hisatake K. Nogi Y. Tsujimoto M. J. Biol. Chem. 2001; 276: 33086-33092Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). RNA was reverse-transcribed by using SuperscriptII reverse transcriptase, 1 mm dNTPs, 1 μg of oligo(dT) primers, and the supplied buffer (Invitrogen). RT-PCR assay was carried out using the following primer pairs: 5′-TGGATGAAATCTCTCGGCGT-3′ (sense) and 5′-TCATGTCTCCCAAGTGGTTC-3′ (antisense). PCR was carried out for 1 cycle at 95 °C for 5 min, followed by 25 cycles at 95 °C for 0.5 min, 57 °C for 0.5 min, and 72 °C for 0.5 min. The relative amounts of the cathepsin K mRNAs were normalized to that of the β-actin mRNA (39Matsumoto M. Sudo T. Maruyama M. Osada H. Tsujimoto M. FEBS Lett. 2000; 486: 23-28Crossref PubMed Scopus (76) Google Scholar). Plasmid Construction—The pcDNA3NFATc1 plasmid was kindly provided by Dr. M. A. Brown (Emory University School of Medicine). The pcDNA3PU.1 plasmid was a kind gift of Dr. H. Sighn (Harvard Medical School). The MITF expression plasmid, pcDNAFLAG-MITF, was constructed as described previously (38Matsumoto M. Hisatake K. Nogi Y. Tsujimoto M. J. Biol. Chem. 2001; 276: 33086-33092Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The expression plasmids for murine p38, pcDNA3p38, and the constitutive active form of MKK6 (MKK6CA), pSRαMKK6, were kindly provided by Dr. T. Sudo (RIKEN) and Dr. M. Karin (University of California, San Diego), respectively. The cathepsin K gene promoter plasmid, p-1108CathepsinLuc, was constructed by obtaining a human genomic clone encoding the 5′-flanking region of the cathepsin K gene by PCR. The fragments of the sequentially deleted sequence from –1108, –818, –516, or –222 to +1 of the cathepsin K gene promoter were amplified by PCR using specific primers as a template. These fragments were then subcloned into MluI/HindIII sites of pGL3 basic vector containing the luciferase reporter gene (Promega, Madison, WI). Transfection and Luciferase Assay—For transfection of the reporter plasmid, RAW264 cells were plated on 12-well plates at a density of 3 × 105 cells/well the day before transfection. A total of 2 μg of plasmid DNA was mixed with Superfect (Qiagen, Santa Clarita, CA) and transfected into the cells following the manufacturer's protocol. After 48 h of transfection, the cells were washed three times with PBS and then lysed in reporter lysis buffer (Promega; Madison, WI). Luciferase activity was then measured with a luciferase assay system (Promega) according to the manufacturer's instructions. Luciferase activity was measured in triplicate, averaged, and then normalized to β-galactosidase activity to correct for transfection efficiency. β-Galactosidase activity was measured by using an o-nitrophenyl-β-d-galactopyranoside as a substrate. Electrophoretic Mobility-shift Analysis (EMSA)—The full-length murine NFATc1 and PU.1 cDNAs were transcribed by using T7 RNA polymerase, and the RNAs were then subjected to in vitro translation with rabbit reticulocyte lysate (Promega) as described previously (40Matsumoto M. Tanaka N. Harada H. Kimura T. Yokochi T. Kitagawa M. Schindler C. Taniguchi T. Biol. Chem. Hoppe-Seyler. 1999; 380: 699-703Crossref PubMed Scopus (94) Google Scholar). Oligonucleotide probes employed were labeled with [γ-32P]ATP using T4 polynucleotide kinase. DNA binding assays were performed by incubating 2 μl of reticulocyte lysates and 32P-labeled double-stranded oligonucleotides for 30 min at 30 °C. For competition assays, a 200-fold excess of unlabeled double-stranded oligonucleotides was added in the reaction mixture. The samples were then resolved on a 4% polyacrylamide gel run in TBE buffer. Western Blot Analysis—Immunoblot analyses and immunoprecipitations were performed as described. In brief, cells were lysed in lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm Na2EDTA, 1 mm EGTA, 1% Triton, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm Na3VO4, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride). Whole cell extracts (WCEs) were prepared by centrifugation at 10,000 × g for 15 min at 4 °C after sonication of cells four times for 5 s. WCEs (30 μg) were electrophoresed on a 12% SDS-polyacrylamide gel and blotted onto a polyvinylidene difluoride membrane. Immunoblot detection was performed with the corresponding antibodies using an ECL detection kit (Amersham Biosciences). Protein Kinase Assays—p38 MAP kinase activity was measured in an immune complex kinase assay. Phosphorylated p38 MAP kinase was immunoprecipitated by anti-phosphorylated p38 (pp38) polyclonal antibody immobilized on agarose beads (anti-pp38 Ab-agarose). After 3 h of incubation at 4 °C, the immunoprecipitates were collected and washed twice with WCEs lysis buffer and then twice with kinase buffer (25 mm Tris, pH 7.5, 5 mm β-glycerophosphate, 2 mm dithiothreitol, 0.1 mm Na3VO4, 10 mm MgCl2). Immunoprecipitates of anti-pp38 Ab-agarose were mixed with 2 μg of GST fused to the truncated form of NFATc1 (GST-NFATc1) protein as a substrate and 5 μCi of [γ-32P]ATP in 30 μl of kinase buffer. The reaction mixture was further incubated for 30 min at 30 °C. The kinase reaction was terminated in the appropriate volume of SDS sample buffer. The phosphorylated GST-NFATc1 was then detected by autoradiography. Immunofluorescence Staining—Authentic osteoclasts on coverslips were fixed in PBS containing 4% paraformaldehyde for 10 min at room temperature and then washed in PBS. The coverslips were permeabilized in 0.1% Triton X-100 for 5 min, immersed in 5% normal goat serum for 30 min, incubated in the appropriate primary antibodies for 1 h, washed in PBS, incubated for 1 h in the appropriate fluorescent-conjugated secondary antibodies, and washed. All coverslips were mounted in OCT compound. Cells were examined by using fluorescence microscopy (Axioplan2 Imaging MOT; Zeiss). Images were recorded, and composite images were compiled by using AxioVision 3.1. The image enhancement was performed by using Adobe Photoshop 6.0. ChIP Assay—The ChIP assay was carried out essentially as described (41Saccani S. Pantano S. Natoli G. J. Exp. Med. 2001; 193: 1351-1359Crossref PubMed Scopus (338) Google Scholar), with a minor modification. Briefly, human PBMC, macrophages, and osteoclasts were treated with 1% formaldehyde for 15 min. The cross-linked chromatin was prepared and sonicated prior to being immunoprecipitated with antibodies against PU.1 and NFATc1 and the acetylated Lys14 (K14) and phosphorylated Ser10 (S10) forms of histone H3 (Upstate Biotechnology Inc., Lake Placid, NY) or control rabbit or murine IgG antibody (Santa Cruz, CA) at 4 °C overnight. After reversal of cross-linking, genomic DNA associated with the immunoprecipitated chromatin was used as a template for amplification by PCR at 27 or 30 cycles by using various sets of primers as indicated below. The amplified PCR product was resolved by 1.5% agarose gel electrophoresis, subsequently transferred to nylon membrane, and subjected to Southern blot analysis with a 32P-labeled cathepsin K promoter-specific probe spanning each region. For PCR amplification of specific regions of the cathepsin K genomic locus, the following primer sets were used: a region containing the PU.1-binding P2 site, sense, 5′-GAGGGTTTATGCATGGAATCCAGC-3′, and antisense, 5′-TCTAGGACAAGTCACATGAGCTTC-3′; a region containing NFATc1-binding N2 site, 5′-CATCCCAGAGGTGAGAGTCAGAC-3′, and antisense, 5′-GGAGCTTACTTTCTCTTTTGGTGAG-3′; and a region of exon 7, sense, 5′-GTGTATTATGATGAAAGCTGCAATAGC-3′, and antisense, 5′-CTGTTTTTAATTATCCAGTGCTTGTTTCC-3′ as a negative control. p38 MAP Kinase Is Essential for the Induction of Cathepsin K Gene by RANKL—MAP kinases are important for transmitting various extracellular signals to the nucleus in diverse biological phenomena including cell differentiation (4Lee Z.H. Kim H.H. Biochem. Biophys. Res. Commun. 2003; 305: 211-214Crossref PubMed Scopus (328) Google Scholar, 42Teitelbaum S.L. Ross F.P. Nat. Rev. Genet. 2003; 4: 638-649Crossref PubMed Scopus (1324) Google Scholar). We reported previously (38Matsumoto M. Hisatake K. Nogi Y. Tsujimoto M. J. Biol. Chem. 2001; 276: 33086-33092Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) that terminal osteoclast differentiation induced by RANKL is accompanied by the increased expression of cathepsin K mRNA. To determine whether the p38 MAP kinase signaling pathway is involved in the RANKL-induced cathepsin K gene expression, we analyzed cathepsin K mRNA by using semiquantitative RT-PCR in the presence or absence of specific kinase inhibitors. As shown in Fig. 1A, murine bone marrow macrophage (BMMϕ) cells were allowed to differentiate into mononuclear or multinucleated tartrate acid phosphatase (TRAP)-positive osteoclasts in the presence of RANKL and M-CSF for 3–5 days, and total RNAs were isolated for PCR analysis. Fig. 1B shows that RANKL treatment of BMMϕ cells increased the expression of cathepsin K mRNA by 19.8- and 28.9-fold at day 3 and 5, respectively, during osteoclast differentiation. When the cells were treated with RANKL in the presence of SB203580, a specific inhibitor of p38 MAP kinase (43Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1981) Google Scholar, 44Young P.R. McLaughlin M.M. Kumar S. Kassis S. Doyle M.L. McNulty D. Gallagher T.F. Fisher S. McDonnell P.C. Carr S.A. Huddleston M.J. Seibel G. Porter T.G. Livi G.P. Adams J.L. Lee J.C. J. Biol. Chem. 1997; 272: 12116-12121Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar, 45Kumar S. Jiang M.S. Adams J.L. Lee J.C. Biochem. Biophys. Res. Commun. 1999; 263: 825-831Crossref PubMed Scopus (228) Google Scholar), a dramatic reduction in the expression of cathepsin K mRNA as well as inhibition of osteoclast differentiation was observed. By contrast, PD98059, a specific inhibitor of MAP kinase kinase for the MAPK/ERK pathway, had no effect on the RANKL-induced expression of cathepsin K mRNA. Similar results were obtained by using the murine monocyte/macrophage cell line, RAW264, which can differentiate into osteoclasts (data not shown). As control, the expression of β-actin mRNA was not affected by the treatment with RANKL in the presence or absence of the inhibitors. These results suggest that the induction of cathepsin K expression by RANKL is largely mediated by the p38 MAP kinase but not the MAPK/ERK signaling pathway. Identification of RANKL-responsive Elements for NFAT Family and PU.1 in Cathepsin K Gene Promoter—Previous analyses of human cathepsin K gene promoter revealed several potentially important regulatory cis-elements, including the PU.1 and E box sites (46Rood J.A. Van Horn S. Drake F.H. Gowen M. Debouck C. Genomics. 1997; 41: 169-176Crossref PubMed Scopus (44) Google Scholar, 47Li Y.P. Chen W. J. Bone Miner. Res. 1999; 14: 487-499Crossref PubMed Scopus (53) Google Scholar). In addition to these regulatory cis-elements, we also found two DNA sequences that closely matched the consensus sequence of the NFAT family (5

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