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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;

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.

Journal of Dental Research, Vol. 82, No. 7, 546-550 (2003)
DOI: 10.1177/154405910308200712


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