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Cot/Tpl2 is Essential for RANKL Induction by Lipid A in Osteoblasts

¹ Laboratory of Host Defense and Germfree Life, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan;
² Department of Periodontology, School of Dentistry, Aichi-Gakuin University, Chikusa-ku, Nagoya, Japan;
³ Research Center for Prevention of Infectious Diseases, Medical Institute of Bioregulation, Kyushu University;
⁴ Department of Molecular Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka, Japan; and
⁵ National Institute for Basic Biology, Okazaki, Japan;

*corresponding author, tmatsugu{at}med.nagoya-u.ac.jp

	ABSTRACT

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Lipopolysaccharide (LPS) is a pathogenic factor that increases bone resorption in periodontal diseases. LPS treatment of osteoblasts was shown to induce the receptor activator of NF-B ligand (RANKL), an essential secretory or membrane-bound factor for osteoclast function, in a manner dependent on extracellular signal-regulated kinase (ERK) activation. However, the mechanisms regulating this process remained unknown. Here, we show that RANKL mRNA induction and ERK activation, when treated with synthetic lipid A (an active center of LPS), were markedly reduced in mouse osteoblasts lacking Cot/Tpl2, which was recently recognized as an essential kinase for the induction of TNF- by LPS in macrophages. In contrast, c-Jun N-terminal kinase (JNK), p38 kinase, Raf-1, and NF-B were normally activated in cot/tpl2-/- osteoblasts. These findings indicate that Cot/Tpl2 is essential for LPS-induced ERK activation and RANKL induction in osteoblasts.

KEY WORDS: Cot • Tpl2 • RANKL • osteoblast • lipid A

	INTRODUCTION

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Receptor activator of NF-B ligand (RANKL), also called osteoclast differentiation factor (ODF) (Yasuda et al., 1998), osteoprotegerin ligand (OPGL) (Lacey et al., 1998), or TNF-related activation-induced cytokine (TRANCE) (Wong et al., 1999), is a cytokine which belongs to the tumor necrosis factor family (Anderson et al., 1997). It plays a major role in homeostasis of the bone by inducing differentiation (Lacey et al., 1998; Malyankar et al., 2000) and biological activity (Lacey et al., 1998) of osteoclasts. It also inhibits apoptosis of osteoclasts (Fuller et al., 1998). These functions of RANKL are mediated by binding to its receptor, RANK (receptor activator of NF-B). Indeed, in the presence of low levels of macrophage-colony-stimulating factor (M-CSF), RANKL appears to be necessary and sufficient for the complete differentiation of osteoclast precursor cells into mature osteoclasts (Lacey et al., 1998; Malyankar et al., 2000). RANKL-deficient mice exhibit severe osteopetrosis and failure in tooth eruption because of a complete absence of osteoclasts and defective bone remodeling (Kong et al., 1999). In addition, several recent reports have clearly indicated that RANKL has several effects on immune cells, including c-Jun N-terminal kinase (JNK) activation and proliferation of T-cells (Wong et al., 1997b), inhibition of apoptosis, and induction of cluster formation of dendritic cells (Wong et al., 1997a).

Periodontal diseases are characterized by the loss of alveolar bone, a process mostly mediated by osteoclasts (Carson et al., 1978). In these diseases, the high local abundance of Gram-negative bacteria-derived lipopolysaccharide (LPS) is responsible, at least in part, for increasing resorption of the bone. Lipid A, a hydrophobic component of LPS, is responsible for most of the biological effects (Schletter et al., 1995). LPS initiates a local host response that involves the generation of cytokines (RANKL, IL-1, TNF-, etc.), chemokines (RANTES, MIP-1, MCP-1, etc.), and prostaglandins (PGs), inducing the recruitment of inflammatory cells (Sweet and Hume, 1996; Kikuchi et al., 2001). It also supports survival and fusion of pre-osteoclasts, leading to bone resorption (Suda et al., 2002). LPS, however, does not directly activate osteoclasts (Suda et al., 2002).

We have previously demonstrated that LPS induces RANKL in osteoblasts, indicating that osteoblasts may mediate the effects of LPS on osteoclasts (Kikuchi et al., 2001). We also showed that an increase in RANKL mRNA by synthetic lipid A seems to be dependent on ERK kinase activation. However, the regulatory mechanism(s) upstream to ERK activation in osteoblasts remained unknown. A serine threonine kinase, Cot (Miyoshi et al., 1991), also known as Tp12 (Patriotis et al., 1993), has recently been recognized as an essential kinase for the induction of TNF- by LPS in mouse macrophages (Dumitru et al., 2000). Interestingly, Cot/Tpl2 is exclusively required for ERK activation in the LPS-treated mouse macrophages, whereas JNK and p38 kinase activation did not require Cot/Tpl2 function (Dumitru et al., 2000).

Here we show that the RANKL mRNA induction and ERK activation, when treated with synthetic lipid A (an active center of LPS), is markedly reduced in the osteoblasts derived from cot/tpl2-/- mice, suggesting that Cot/Tpl2 is essential for LPS-induced ERK activation and RANKL production in osteoblasts. These findings indicate that Cot/Tpl2 could be a possible therapeutic target in the treatment of periodontal diseases.

	MATERIALS & METHODS

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Generation of cot/tpl2-/- Mice
The cot/tpl2 locus was molecularly cloned from the mouse 129/sv genomic DNA library (STRATAGENE, La Jolla, CA, USA). The targeting vector for the cot/tpl2 gene disruption was constructed and introduced into the CCE line of ES cells. Targeted clones were injected into the blastocoel cavity of C57BL/6 embryos, and chimeric mice were back-crossed with C57/BL6 females. Disruption of both cot/tpl2 alleles was confirmed by Southern blot analysis. Absence of cot/tpl2 mRNA and its gene products in the cot/tpl2-/- mice was confirmed by Northern blot and Western blot analyses, respectively. All animal work was approved and performed under the guidelines set by the Nagoya University Institutional Animal Care and Use Committee.

Cells
Osteoblastic cells from ddy mice, a mouse strain occasionally used for osteoblast study, cot/tpl2-/- mice, and their control littermates were isolated from the calvaria of two-day-old fetal mice as described previously (Takahashi et al., 1988). Six calvaria were collected and applied to 5 routine sequential digestions, with use of a solution of -minimum essential medium (-MEM) containing 0.1% collagenase (Wako Pure Chemical Industries, Osaka, Japan) and 0.2% dispase (Godo Shusei, Tokyo, Japan). Cells isolated in fraction 4 were combined and cultured for 7 days in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS (Sigma Chemical Co., St. Louis, MO, USA) at 37°C in 5% carbon dioxide/95% air.

Reagents
Synthetic E. coli-type lipid A, ONO4007, was kindly provided by Ono Pharmaceutical Company (Tokyo, Japan) and previously described (Kikuchi et al., 2001). Anti-phospho-JNK mAb, anti-phospho-ERK mAb, anti-ERK polyclonal Ab, anti-phospho-p38 polyclonal Ab, and anti-phospho-Raf-1 polyclonal Ab were obtained from New England Biolabs (Beverly, MA, USA). Anti-JNK1 polyclonal Ab, anti-p38 polyclonal Ab, anti-Raf-1 polyclonal Ab, and anti-cot/tpl2 polyclonal Ab were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Northern Blot Analysis
Total cellular RNA was extracted from each cell culture with the use of TRIZOL reagent (Gibco BRL, Rockville, MD, USA), according to the manufacturer’s instructions. For RNA blotting, 10-µg aliquots of the total RNA were electrophoresed in a 1% agarose gel containing 20 mmol/L morpholinopropane sulfonic acid (MOPS), 5 mmol/L sodium acetate, 1 mmol/L EDTA (ethylenediaminetetra-acetic acid, pH 7.0), and 6% (vol/vol) formaldehyde. Equal loading of the aliquots was assessed by ethidium bromide staining, and RNAs were transferred to a nylon membrane. After ultraviolet-crosslinking, membranes were soaked in pre-hybridization solution (6X SSC, 5X Denhardt’s reagents, 0.5% SDS, 100 µg/mL denatured salmon sperm DNA, and 50% formamide) for 2 hrs at 65°C, followed by incubation with ³²P-labeled probe in the pre-hybridizaion solution for 14 hrs at 65°C. The membranes were washed twice in 2X SSC and 0.1% SDS for 5 min at 65°C, washed twice in 0.1X SSC and 0.1% SDS for 15 min, and then exposed to films (Fuji RX-U films; Fuji Film, Minamiashigara, Japan).

Extract Preparation and Immunoblotting
Cells were subjected to lysis in RIPA lysis buffer (150 mmol/L NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM Tris-HCl [pH 8.0], 2 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mg/mL sodium orthovanadate, 1 mmol/L phenylmethanesulfonyl fluoride) at 10⁸ cells/mL. The lysates were separated on SDS-polyacrylamide gels, then electro-transferred to polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA, USA). The membranes were blocked for 1 hr in 5% non-fat dry milk/TBST (20 mmol/L Tris-HCl [pH 7.6], 0.15 mol/L NaCl, 0.1% Tween 20), incubated with primary antibody in TBST for 15 hrs, washed 3 times with TBST, and incubated for 1 hr with horseradish peroxidase-conjugated anti-mouse IgG (Amersham Pharmacia Biotech, Piscataway, NJ, USA) diluted 1:10,000 in TBST. After 3 washes in TBST, the blots were developed with the enhanced chemiluminescence system (Amersham Biosciences Corp., Piscataway, NJ, USA), according to the manufacturer’s instructions.

Immune Complex Kinase Assay
Cell lysates (10⁷ cells/sample) were incubated with 0.4 µg of anti-ERK polyclonal Ab for 2 hrs at 4°C, followed by incubation with protein A-Sepharose beads for an additional 1 hr. The beads were washed three times in PLC lysis buffer and then once in kinase buffer (20 mM Tris-HCl [pH 7.4], 20 mM MgCl2, 2 mM EGTA, 0.5 mM sodium vanadate, 10 mM ß-glycerophosphate, and 1 mm dithiothreitol). The kinase reaction was initiated by the addition of 40 µL of kinase buffer with 20 µM ATP, 5 µCi of [³²P]ATP, and 0.5 µg of myelin basic protein (Sigma Chemical Co.), and was allowed to proceed for 20 min at 30°C. The reaction was terminated by the addition of 2X SDS sample buffer. Samples were boiled and resolved by SDS-polyacrylamide gel electrophoresis, and the fixed gel was exposed to an x-ray film.

	RESULTS

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The Expression of cot/tpl2 in Primary Mouse Osteoblasts
We have previously shown that an increase in RANKL mRNA by synthetic lipid A or LPS in osteoblasts seemed to be dependent on ERK kinase activity (Kikuchi et al., 2001). However, it was not clear how LPS activates ERK in osteoblasts. A recent report indicated cot/tpl2 as an essential factor for ERK activation in the LPS-treated macrophages (Dumitru et al., 2000). Thus, we sought to examine if cot/tpl2 is involved in the induction of RANKL mRNA by synthetic lipid A in osteoblasts. First, we investigated the expression of cot/tpl2 in mouse osteoblasts by Western blot analysis. While a small amount of cot/tpl2 was expressed in unstimulated osteoblasts, synthetic lipid A treatment significantly increased the cot/tpl2 protein levels (Fig. 1).

View larger version (16K): [in this window] [in a new window]   Figure 1. Expression of cot/tpl2 in primary mouse osteoblasts. Osteoblasts from calvaria of ddy mice were treated with 1 µg/mL synthetic lipid A for the indicated times. Cell lysates were prepared, and the amount of the cot/tpl2 protein was determined by Western blot analysis with the use of anti-cot/tpl2 polyclonal antibody. Two forms of the cot/tpl2 protein were translated from the alternative initiation codons (Aoki et al., 1991; Miyoshi et al., 1991).

View larger version (44K): [in this window] [in a new window]   Figure 2. Impairment of synthetic lipid-A-mediated RANKL mRNA up-regulation in osteoblasts from cot/tpl2-/- mice. Osteoblasts isolated from calvaria of the wild-type and cot/tpl2-/- mice were treated with indicated doses of synthetic lipid A. At 2 hrs after the treatment, total RNA was extracted, separated in a 1% agarose/formaldehyde gel, and transferred to a nylon membrane. The gene expression of RANKL was analyzed by hybridization with the use of a 32P-labeled specific RANKL cDNA probe. The 18S and 28S ribosomal RNA bands of the ethidium-bromide-stained gel are shown as a control for equal loading.

View larger version (62K): [in this window] [in a new window]   Figure 3. Activation of ERK by synthetic lipid A is dependent on Cot/Tpl2. (A) Osteoblasts isolated from calvaria of the wild-type and cot/tpl2-/- mice were treated with 1 µg/mL synthetic lipid A for the indicated times. ERK phosphorylation was measured by Western blot with an mAb specific for the phosphorylated form of ERK. The same filters were re-blotted with an anti-ERK antibody to show consistent amounts of the kinase. (B) Osteoblasts isolated from the wild-type and cot/pl2-/- mice were treated with 1 µg/mL synthetic lipid A for the indicated times. Synthetic lipid-A-mediated ERK kinase activation was measured by the in vitro kinase assay with myelin basic protein (MBP) as a substrate. (C) Osteoblasts isolated from the wild-type and cot/tpl2-/- mice were treated with 1 µg/mL synthetic lipid A for the indicated times. Phosphorylation of p38 and JNK was measured by Western blot with the use of Abs specific for the phosphorylated forms of p38 and JNK, respectively. The same filters were re-blotted with an anti-p38 or JNK1 Ab to show consistent amounts of the kinases. Slightly increased basal p38 phosphorylation in cot/tpl2+/+ mice in Fig. 3C was not consistently observed in repeated experiments.

View larger version (18K): [in this window] [in a new window]   Figure 4. Synthetic lipid A induces Raf activation in the cot/tpl2-/- osteoblasts. Osteoblasts isolated from the wild-type and cot/tpl2-/- mice were treated with 1 µg/mL synthetic lipid A for the indicated times. Raf-1 phosphorylation was analyzed with phospho-specific anti-Raf-1 antibody. The same filter was re-probed with an anti-Raf-1 Ab to show consistent amounts of the whole Raf-1 kinase.

	DISCUSSION

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The cot/tpl2 proto-oncogene, originally identified and molecularly cloned by focus-forming assay with the use of SHOK cells (Higashi et al., 1990; Miyoshi et al., 1991), encodes a serine threonine protein kinase (Patriotis et al., 1993). Earlier studies revealed that overexpression of cot/tpl2 activates all three members of MAPK: ERK, JNK, and p38 (Patriotis et al., 1994; Salmeron et al., 1996; Chiariello et al., 2000). cot/tpl2 also regulates the proteolysis of NF-B-inhibitory protein NF-B1 p105, indicating that it may also play an important role in NF-B activation (Belich et al., 1999). A study on cot/tpl2-deficient mice showed that cot/tpl2 is required for the activation of ERK1 and ERK2 by LPS in mouse macrophages, and that they are obligatory for the post-transcriptional regulation of TNF- (Dumitru et al., 2000). Macrophages from cot/tpl2-/- mice, however, showed normal responses of p38, JNK, and NF-B activation to LPS. The report also showed that cot/tpl2-/- mice are resistant to LPS/D-Galactosamine-induced endotoxin shock, and that their resistance is due to defective post-transcriptional regulation of TNF- expression in macrophages. Thus, cot/tpl2 is essential for ERK activation and TNF- production in macrophages, but its role in other signaling pathways remains somewhat controversial. Besides, roles of cot/tpl2 in cell types other than macrophages have not been extensively studied.

We have previously reported that LPS and synthetic lipid A potently increased RANKL mRNA expression in mouse osteoblasts derived from calvaria, whereas PD98059, a specific inhibitor of MEK, effectively inhibited this process, indicating that RANKL mRNA induction by LPS depends on ERK activation (Kikuchi et al., 2001). Analysis of our current data demonstrated that the synthetic lipid-A-mediated RANKL mRNA up-regulation and ERK activation (Fig. 3A) are severely impaired in cot/tpl2-/- osteoblasts (Fig. 2). In contrast, they showed normal activation of p38, JNK, and NF-B in response to synthetic lipid A (Fig. 3B; data not shown). Our result is consistent with the previous findings on macrophages and indicates that cot/tpl2 is essential for ERK activation, but not for p38, JNK, and NF-B activation, after LPS stimulation in at least two cell types: macrophages and osteoblasts.

Notably, analysis of our data indicated that Raf-1 activation is not sufficient to activate ERK in mouse osteoblasts (Fig. 4). The early descriptions on ERK activation showed that ligation of many receptors led to activation of Ras-Raf-1-MEK-ERK activation (Widmann et al., 1999). The role of Raf-1 in LPS-mediated ERK activation has not been well-established. In fact, LPS activates ERK without Raf-1 activation in RAW264.7 cells (Guthridge et al., 1997) and alveolar macrophages (Monick et al., 2000). At present, we are unable to explain why Raf-1 activation does not lead to ERK activation in mouse osteoblasts. Raf-1 may require Cot/Tp12 to activate ERK in osteoblasts, because cot/tpl2 has been reported to act in concert with Raf-1 to activate ERK in macrophages (Patriotis et al., 1994).

The promoter region of the mouse RANKL gene contains inverted CAAT boxes and a putative Cbfa1/Osf2/AML3 binding domain with no obvious NF-B binding motifs (Kitazawa et al., 1999). Since both CAAT/enhancer binding protein (C/EBP) and Cbfa1 are activated by ERKs (Xiao et al., 2000), it is conceivable that the cot/tpl2-ERK pathway is important for the RANKL mRNA induction by synthetic lipid A through one or both of these factors. Interestingly, the molecular mechanism(s) of the cot/tpl2-ERK pathway as a regulator of RANKL mRNA transcription in osteoblasts are in sharp contrast to those of TNF- production in macrophages. In macrophages, cot/tpl2 is not required for TNF- mRNA transcription, but seems to be essential for the proper transport of the mRNA for protein translation.

Taken together, the RANKL mRNA induction in response to synthetic lipid A depends on the cot/tpl2-transduced ERK activation signals. Since LPS-mediated RANKL production by osteoblasts induces differentiation of osteoclasts, which is a major impairing factor of periodontitis, these findings may provide an insight into a therapeutic approach to controlling the disease.

	ACKNOWLEDGMENTS

 
We thank Ms. K. Itano, Ms. H. Yamaguchi, and Ms. A. Nishikawa for their technical assistance. This work was supported by grants from Ono Pharmaceutical Company and the Ministry of Education, Science and Culture of the Japanese Government.

Received October 1, 2002; Last revision February 17, 2003; Accepted April 8, 2003

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