Osteoprotegerin differentially regulates protease expression in osteoclast cultures Y.Wittrant, a S.Couillaud, a S.Theoleyre, a C.Dunstan, b D.Heymann, a andF.R edini a, * a Facult e deM edecine, Laboratoire de Physiopathologie de la R esorption Osseuse, 1 rue Gaston Veil, 44035 Nantes cedex 01, France b Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA Received14March2002 Abstract Cysteineproteasesandmatrixmetalloproteinases(MMPs)areimportantfactorsinthedegradationoforganicmatrixcompo- nentsofbone.Osteoprotegerin(OPG)isanosteoblast-secreteddecoyreceptorthatinhibitsosteoclastdifferentiationandactivation. This study investigated the direct effects of human OPG on cathepsin K, MMP-9, MMP-2, and tissue inhibitors of metallopro- teinases(TIMP1andTIMP2)expressedbypurifiedrabbitosteoclasts.Theexpressionoftwoosteoclastmarkers,namelytartrate- resistantacidphosphatase(TRAP)andcathepsinK,wasinhibitedby100ng/mLhOPG,whereasMMP-9expressionwasenhanced. Gelatinaseactivitiesweremeasuredusingazymographicassay,andhOPGwasshowntoenhancebothpro-MMP-9andMMP-2 activities.Concomitantly,TIMP1expressionwasgreatlystimulatedbyhOPG,whereasTIMP2mRNAlevelswerenotmodulated. Overall,theseresultsshowthathOPGregulatestheproteasesproducedbypurifiedosteoclastsdifferentially,producingamarked inhibitoryeffectontheexpressionofcathepsinK,themainenzymeinvolvedinboneresorption. Ó 2002ElsevierScience(USA).All rightsreserved. Keywords: Osteoclast;Boneresorption;Osteoprotegerin;Metalloproteinases;Tissueinhibitorsofmetalloproteinases;CathepsinK Osteoclastsarelargemultinucleatedcellsoriginating from bone marrow and are involved in bone deminer- alization and resorption. In normal bone physiology, resorption,matrixsynthesis,andmineralizationareas- sociated processes that appear to be dysregulated in various metabolic bone diseases, resulting in excessive bone loss (osteoporosis, osteolytic bone tumours) or excessiveboneformation(osteopetrosis,osteosclerosis). The key substances regulating osteoclast differentia- tion and activation have recently been identified. Re- ceptoractivatorofNF-jBligand(RANKL),amember ofthetumournecrosisfactor(TNF)cytokinefamily,is a downstream regulator of osteoclast formation and activation, inducing many hormones and cytokines to produce osteoresorptive effects [1]. Within the bone system,RANKLisexpressedonosteoblastlineagecells andexertsitsbiologicaleffectbybindingtotheRANK receptor at the surface of osteoclasts [2]. The third protagonist, osteoprotegerin (OPG), is produced by osteoblastic/stromal cells and acts as a decoy receptor for RANKL, preventing it from binding to and acti- vating RANK on the osteoclast surface [3]. Thus, the biological effects of OPG on bone cells include the in- hibition of terminal stages of osteoclast differentiation, suppressionoftheactivationofmatureosteoclasts,and inductionofapoptosis. Once activated, the osteoclasts secrete both protons and proteinases at their attachment site, resulting in dissolution of bone mineral and degradation of the matrix(mainlycomposedoftypeIcollagen)[4].Several studiesindicatethattheproteinasesinvolvedinsolubi- lizationofthiscollagenousmatrixbelongtothecysteine proteinaseandmatrixmetalloproteinase(MMP)groups [5,6].TheidentityofthespecificMMPsresponsiblefor collagendegradationintheresorptionzoneisunknown, although some MMPs have been immunolocalized in this resorption compartment [7]. It seems that MMP-9 atleast,whichisknowntobethemostabundantgela- tinolytic MMP in osteoclasts, is not rate-limiting [8]. The zinc-dependent endopeptidase activities of the BiochemicalandBiophysicalResearchCommunications293(2002)38–44 www.academicpress.com * Correspondingauthor.Fax:+33-2-40-41-28-60. E-mail address: francoise.redini@sante.univ-nantes.fr(F.R edini). 0006-291X/02/$-seefrontmatter Ó 2002ElsevierScience(USA).Allrightsreserved. PII:S0006-291X(02)00179-1