Regulation of osteoclast protease expression by RANKL q Y. Wittrant, S. Theoleyre, S. Couillaud, C. Dunstan, 1 D. Heymann, and F. R edini * Laboratoire de Physiopathologie de la R esorption Osseuse et Th erapie des Tumeurs Osseuses Primitives EE 99-01, Facult e de M edecine, 1 rue Gaston Veil, 44035 Nantes cedex 1, France Received 4 September 2003 Abstract Receptor activator of NF-jB ligand (RANKL) is essential for osteoclast (OC) differentiation/activation and functions through its receptor RANK at the surface of the osteoclastic cells. This study investigated for the first time the direct effects of hRANKL on protease/protease inhibitor expressions and protease activities in purified rabbit osteoclast cultures, using semi-quantitative RT- PCR, gelatin zymography, and enzymatic assays. RANKL was shown to exert in vitro pro-resorptive effects by increasing osteoclast marker expressions (Tartrate resistant acid phosphatase (TRAP) and cathepsin K), MMP-9 expression, and pro-MMP-9 activity and by diminishing TIMP-1 expression, leading to an up-regulation of the MMP-9/TIMP-1 ratio. Ó 2003 Elsevier Inc. All rights reserved. Keywords: RANKL; Osteoclasts; Bone resorption; Metalloproteinase Bone remodeling is a physiological process that in- volves the resorption of bone by osteoclasts and the synthesis of bone matrix by osteoblasts. Osteoclasts are specialized monocyte/macrophage family members that differentiate from hematopoietic precursors [1]. Re- cently, a new tumor necrosis factor-family molecule named Receptor activator of NF-jB ligand (RANKL) has been shown to both activate mature osteoclasts and mediate osteoclastogenesis in the presence of M-CSF [2,3]. RANKL is preferentially expressed on committed preosteoblastic cells, whereas its specific receptor RANK is expressed in hematopoietic osteoclast pro- genitors [4,5]. RANKL knockout mice have severe os- teopetrosis and defective tooth eruption [6]. Thus, RANKL is the key osteoclast differentiation factor ab- solutely required for osteoclast development and bone modeling in vivo, and also functions as a survival factor for osteoclast precursors. Proteinases play an important role in bone physiol- ogy not only because they are required for the solubili- zation of bone matrix, but also because they are key components of the mechanisms that determine where and when bone resorption will be initiated. Cysteine proteinases and matrix metalloproteinases (MMPs) have been identified as the main proteinases active in these processes. Among cysteine proteinases, cathepsin K plays an essential role in osteoclast-mediated degra- dation of bone organic matrix [7]. Knockout of the en- zyme in mice, as well as lack of functional enzyme in the human pathological condition pycnodysostosis, results in osteopetrosis [8,9]. Among MMPs, MMP-9 is known as the most abundant gelatinolytic MMP in osteoclasts but is not rate limiting, since the resorbing activity of the osteoclasts in bone explants from MMP-9-null mice compares to that of wild-type mice [10]. MMP-9 was shown to be the main MMP involved in the invasive activity of osteoclasts [11]. The zinc-dependent endo- peptidase activities of the MMP-9 and -2 are inhibited specifically by their intrinsic inhibitors called tissue inhibitors of metalloproteinases (TIMP)-1 and -2, respectively [12]. We previously demonstrated that osteoprotegerin (OPG) exerts differential effects on protease activities in osteoclast cultures [13], but a unique study reported the q Abbreviations: MMP, matrix metalloproteinase; OC, osteoclast; RANK, receptor activator of NF-jB; RANKL, RANK ligand; TRAP, tartrate resistant acid phosphatase; TIMP, tissue inhibitors of MMP. * Corresponding author. Fax: +1-33-2-40-41-28-60. E-mail address: francoise.redini@sante.univ-nantes.fr (F. R edini). 1 Amgen, One Amgen Center Drive, Thousand Oaks, CA 91320, USA. 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.09.084 Biochemical and Biophysical Research Communications 310 (2003) 774–778 BBRC www.elsevier.com/locate/ybbrc