Family 2 cystatins inhibit osteoclast-mediated bone resorption in calvarial bone explants H.S. Brand, a, * U.H. Lerner, b A. Grubb, c W. Beertsen, d A.V. Nieuw Amerongen, a and V. Everts e,f a Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), Universiteit van Amsterdam and Vrije Universiteit, Amsterdam, The Netherlands b Department of Oral Cell Biology, Umea University, Umea, Sweden c Department of Clinical Chemistry, University of Lund, Sweden d Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), Universiteit van Amsterdam and Vrije Universiteit, Amsterdam, The Netherlands e Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), Universiteit van Amsterdam and Vrije Universiteit, Amsterdam, The Netherlands f Department of Cell Biology and Histology, Academic Medical Centre, Amsterdam, The Netherlands Received 1 March 2004; revised 12 May 2004; accepted 14 May 2004 Available online 4 July 2004 Abstract Osteoclastic bone resorption depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Biochemical studies have shown that cystatins, naturally occurring inhibitors of these enzymes, inhibit bone matrix degradation. Since the mechanism by which cystatins exert this inhibitory effect is not completely resolved yet, we studied the effect of cystatins on bone resorption microscopically and by Ca-release measurements. Calvarial bone explants were cultured in the presence or absence of family 2 cystatins and processed for light and electron microscopic analysis, and the culture media were analyzed for calcium release. Both egg white cystatin and human cystatin C decreased calcium release into the medium significantly. Microscopic analyses of the bone explants demonstrated that in the presence of either inhibitor, a high percentage of osteoclasts was associated with demineralized non-degraded bone matrix. Following a 24-h incubation in the presence of cystatin C, 41% of the cells were adjacent to areas of demineralized non-degraded bone matrix, whereas in controls, this was only 6%. If bone explants were cultured with both PTH and cystatin C, 60% of the osteoclasts were associated with demineralized non-degraded bone matrix, compared to 27% for bones treated with PTH only (P < 0.01). Our study provides evidence that cystatins, the naturally occurring inhibitors of cysteine proteinases, reversibly inhibit bone matrix degradation in the resorption lacunae adjacent to osteoclasts. These findings suggest the involvement of cystatins in the modulation of osteoclastic bone degradation. D 2004 Elsevier Inc. All rights reserved. Keywords: Bone; Calcium; Cathepsins; Cystatins; Cysteine proteinases; Osteoclasts Introduction Skeletal remodeling is a highly regulated process that involves both the formation and resorption of bone. Degra- dation of bone is accomplished by the multinucleated osteoclast. This cell attaches to the bone surface, seals off an area from the extracellular environment, and degrades the bone in the center of this segregated site. The resorption site is characterized by deep invaginations of the osteoclast membrane, the ruffled border. Using a cell surface H+- ATPase, the osteoclast acidifies the ruffled border area. This solubilizes the calcium hydroxyapatite crystals from the bone and also creates an optimal environment for the activity of the acidic proteolytic enzymes that digest the bone matrix [1–10]. Several lines of evidence indicate the crucial role of cysteine proteinases in osteoclastic bone digestion. Cysteine proteinase inhibitors, for example, E-64, leupeptin, and Z- Phe-Ala-CHN2 reduce osteoclastic pit formation in tissue 8756-3282/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2004.05.015 * Corresponding author. Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. Fax: +31-20-444-8685. E-mail address: hs.brand@vumc.nl (H.S. Brand). www.elsevier.com/locate/bone Bone 35 (2004) 689 – 696