Previously, we reported two types of neutral ceramidase in mice, one solubilized by freeze-thawing and one not. The former was purified as a 94-kDa protein from mouse liver, and cloned (Tani, M., Okino, N., Mori, K., Tanigawa, T., Izu, H., and Ito, M. (2000) J. Biol. Chem. 275, 11229 -11234). In this paper, we describe the purification, molecular cloning, and subcellular distribution of a 112-kDa membrane-bound neutral ceramidase of rat kidney, which was completely insoluble by freeze-thawing. The open reading frame of the enzyme encoded a polypeptide of 761 amino acids having nine putative N-glycosylation sites and one possible transmembrane domain. In the ceramidase overexpressing HEK293 cells, 133-kDa (Golgi-form) and 113-kDa (endoplasmic reticulum-form) Myc-tagged ceramidases were detected, whereas these two proteins were converted to a 87-kDa protein concomitantly with loss of activity when expressed in the presence of tunicamycin, indicating that the N-glycosylation process is indispensable for the expression of the enzyme activity. Immunohistochemical analysis clearly showed that the ceramidase was mainly localized at the apical membrane of proximal tubules, distal tubules, and collecting ducts in rat kidney, while in liver the enzyme was distributed with endosome-like organelles in hepatocytes. Interestingly, the kidney ceramidase was found to be enriched in the raft microdomains with cholesterol and GM1 ganglioside.Over the past decade, sphingolipids and their metabolites have emerged as a new class of lipid biomodulators of various cell functions (1, 2). Ceramide (N-acylsphingosine; Cer), 1 a common lipid backbone of sphingolipids, functions as a second messenger in a variety of cellular events including apoptosis and cell differentiation (3, 4). Sphingosine (Sph) has bifunctional effects on cell growth, i.e. it exerts mitogenic (5) and apoptosis inducing (6) activities, depending on the cell type and cell cycle. Sph-1-phosphate (S1P) was found to function as an intra-and intercellular second messenger to regulate cell growth (7), motility (8), and morphology (9). Interestingly, S1P inhibits the apoptosis induced by Cer and Fas ligand (10), indicating that the balance of Cer/Sph/S1P affects cell phenotype. Ceramidase (CDase, EC 3.5.1.23) is an enzyme that catalyzes hydrolysis of the N-acyl linkage of Cer to produce Sph, which can be phosphorylated to S1P by sphingosine kinase (11). Sph is not produced by de novo synthesis (12), and thus the activity of CDase is crucial not only for switching off the Cer-induced signaling but also for generation of Sph and S1P. CDase is classified into two categories: acid and neutral/alkaline enzymes depending on pH optimum. Acid CDase is thought to be a housekeeping enzyme to catabolize Cer in lysosomes. The enzyme was purified from human urine (13), and cDNA encoding the enzyme was isolated from cDNA libraries of human (14) and mouse (15). A deficiency of the enzyme could cause Farber disease in which Cer is accumulated in lysosomes (16). Neutral/alkaline CDase seems...

Osteoporosis remains a major public health problem through its associated fragility fractures. Several animal models for the study of osteoporotic bone loss, such as ovariectomy (OVX) and denervation, require surgical skills and several weeks to establish. Osteoclast differentiation and activation is mediated by RANKL. Here we report the establishment of a novel and rapid bone loss model by the administration of soluble RANKL (sRANKL) to mice. Mice were injected intraperitoneally with sRANKL and used to evaluate existing anti-osteoporosis drugs. sRANKL decreased BMD within 50 h in a dose-dependent manner. The marked decrease in femoral trabecular BMD shown by pQCT and the 3D images obtained by mCT were indistinguishable from those observed in the OVX model. Histomorphometry showed that osteoclastic activity was significantly increased in the sRANKL-injected mice. In addition, serum biochemical markers of bone turnover such as Ca, C-telopeptide of type 1 collagen (CTX), and TRACP5b were also significantly increased in the sRANKL-injected mice in a dose-dependent manner. Bisphosphonates (BPs), selective estrogen receptor modulators (SERMs), and PTH are commonly used for the treatment of osteoporosis. We successfully evaluated the effects of anti-bone-resorbing agents such as BPs, a SERM, and anti-RANKL-neutralizing antibody on bone resorption in a couple of weeks. We also evaluated the effects of PTH on bone formation in 2 wk. A combination of sRANKL injections and OVX made it possible to evaluate a SERM. The sRANKL model is the simplest, fastest, and easiest of all osteoporosis models and could be useful in the evaluation of drug candidates for osteoporosis.

We report here the molecular cloning, sequencing, and expression of the gene encoding the mouse neutral ceramidase, which has been proposed to function in sphingolipid signaling. A full-length cDNA encoding the neutral ceramidase was cloned from a cDNA library of mouse liver using the partial amino acid sequences of the purified mouse liver ceramidase. The open reading frame of 2,268 nucleotides encoded a polypeptide of 756 amino acids having nine putative N-glycosylation sites. Northern blot analysis revealed that the mRNA of the ceramidase was expressed widely in mouse tissues, with especially strong signals found in the liver and kidney. The ceramidase activity of lysates of CHOP cells increased more than 900-fold when the cells were transformed with a plasmid containing the cDNA encoding ceramidase. We also cloned the ceramidase homologue from the cDNA library of mouse brain and found that the sequence of the open reading frame, but not the 5-noncoding region, was identical to that of the liver.

Background: A RANKL-binding peptide WP9QY (W9) is known to inhibit osteoclastogenesis.Results: W9 showed an anabolic effect on cortical bone in mice. W9 bound RANKL and differentiated osteoblasts with production of autocrine factors like BMP-4.Conclusion: Signaling through RANKL is involved in part in the W9-induced osteoblast differentiation.Significance: The RANKL pathway could be a novel mechanism in osteoblast differentiation.

Background: Receptor activator of nuclear factor-κB ligand is a pivotal osteoclast differentiation factor.Results: Daily injection of parathyroid hormone increased bone mass by stimulating bone formation in the anti-receptor activator of nuclear factor-κB ligand antibody-treated mice.Conclusion: Parathyroid hormone exerted its bone anabolic activity in mice with few osteoclasts.Significance: Parathyroid hormone requires no osteoclasts for stimulating bone formation.