The Regulation of Cathepsin K Gene Expression

The Regulation of Cathepsin K Gene Expression BRUCE R. TROEN Geriatrics Research, Education, and Clinical Center & Research Service, Miami Veterans Affairs Medical Center, and Geriatrics Institute, Department of Medicine, Miller School of Medicine, University of Miami, Miami, Florida 33125, USA ABSTRACT: Cathepsin K is essential for normal bone resorption. Osteoclasts synthesize and secrete cathepsin K into the extracellular compartment at the attachment site between osteoclasts and the bone surface, wherein the organic matrix is subsequently degraded by cathepsin K. RANKL, NFAT, Mitf, and various components of AP-1 enhance osteoclast formation and bone resorption, whereas IFN-, calcitonin, estradiol, and calcium inhibit it. These agents appear to act correspondingly to alter cathepsin K mrna and protein expression in order to stimulate and suppress the osteoclast s resorbing potential. RANKL signaling via the calcineurin-calcium-nfat signaling cascade plays a significant role in the regulation of cathepsin K expression. Activation via p38 and the micropthalmia transcription factor also enhances cathepsin K expression. Future studies will be needed to elucidate the relative roles of various signaling pathways at different stages of osteoclast formation and activation and to determine whether genetically disrupting these pathways can modulate bone resorption with or without impeding other osteoclast functions. KEYWORDS: cathepsin K; osteoclast; RANK ligand; NFAT; bone resorption INTRODUCTION Osteoporosis is one of the leading causes of morbidity in the elderly, resulting in over 1 million fractures each year and increased mortality for those with hip fractures. Osteoporosis is characterized by a progressive loss of total bone mass and bone density due to an imbalance between osteoclastic bone resorption and osteoblastic bone formation. Normal bone resorption and remodeling critically depend upon the synthesis and secretion of cathepsin K by osteoclasts (for reviews, please see Refs. 1 3). Humans with mutations in the cathepsin Address for correspondence: Bruce R. Troen, M.D., Miami VA Medical Center, 11 GRC, 1201 NW 16th Street, Miami, FL 33125. Voice: 305-575-3388; fax: 305-575-3365. e-mail: troen@miami.edu Ann. N.Y. Acad. Sci. 1068: 165 172 (2006). C 2006 New York Academy of Sciences. doi: 10.1196/annals.1346.018 165

166 ANNALS NEW YORK ACADEMY OF SCIENCES K (CTSK) gene exhibit skeletal abnormalities (pycnodysostosis), and mice that are completely CTSK-deficient develop osteopetrosis. Therefore, CTSK plays an important role in the development of osteoporosis. Indeed, a number of pharmaceutical firms are actively developing inhibitors of cathepsin K enzymatic activity and are conducting clinical trials to use these agents as an antiresorptive to treat osteoporosis. Despite these clinically translational studies, the regulation of CTSK expression during osteoclast differentiation and in active bone-resorbing osteoclasts is incompletely understood. However, in the past several years, an increasing number of studies have begun to delineate the mechanisms underlying the transcription of the CTSK gene. Here we briefly review the progress made in understanding the regulation of CTSK gene expression. REGULATION OF CTSK EXPRESSION The regulation of bone resorption involves a complicated set of hormonal and/or cytokine interactions that initially stimulate osteoblasts and stromal cells, which then elaborate factors that signal osteoclasts to degrade bone (for reviews see Refs. 1,4 6). Key to understanding the regulation of cathepsin K expression is that many of the agents that have been shown to induce osteoclast formation and activation or to inhibit osteoclast activity enhance and suppress, respectively, cathepsin K gene expression. The receptor activator of NF- B ligand (RANKL) plays a critical role in osteoclast differentiation and activation. 7 9 RANKL is a membrane-bound factor that is produced by osteoblasts and stromal cells in response to a variety of signals. RANKL binds to the cytoplasmic membrane receptor RANK (receptor activator of NF- B), which is a member of the tumor necrosis factor (TNF) receptor superfamily, and subsequently induces both osteoclast differentiation and activation. RANKL stimulates CTSK mrna and protein expression in human osteoclasts. 10 We have demonstrated that RANKL acutely stimulates cathepsin K mrna expression in isolated mature rat osteoclasts. 11 In addition, as bone marrow-derived osteoclasts differentiate and fuse, they produce increasing amounts of cathepsin K. 11 RANKL also enhances CTSK mrna expression in murine myeloid RAW 264.7 cells concomitant with induction toward an osteoclastic phenotype. 12 This phenomenon is both concentration- and time-dependent over a 7-day period 13 and also occurs at earlier times. 14 TNF-, which is a member of the same superfamily of ligands as RANKL, also stimulates CTSK mrna expression in pre-osteoclasts. 15 A large number of agents regulate the production of RANKL by osteoblasts and stromal cells. 1 Stimulators include vitamin D, parathyroid hormone, TNF-, glucocorticoids, interleukins 1 (IL-1) and 11 (IL-11), thyroid hormone, prostaglandin E2, lipopolysaccharide, fibroblast growth factor-2, histamine, insulin-like growth factor-1, histamine, and low gravity. Inhibitors of RANKL expression include estro-

TROEN: REGULATION OF CATHEPSIN K GENE EXPRESSION 167 gen and transforming growth factor-. Consequently, these agents indirectly exert corresponding stimulatory and inhibitory effects upon cathepsin K expression. RANKL appears to stimulate the transcription of the cathepsin K gene via a number of mechanisms. An early and proximal event in RANKL-mediated signaling involves activation of TRAF6 (TNF receptor-associated factor 6), which is a critical adaptor molecule for the cognate receptor of RANKL. Overexpression of TRAF6 stimulates cathepsin K promotor activity, and RANKL stimulation of cathepsin K promotor activity is inhibited by the overexpression of dominant negative TRAF6. 14 More distally in the signaling pathway, Matsumoto et al. demonstrated that RANKL leads to the phosphorylation of NFAT2 by p38, thereby inducing translocation of NFAT2 into the nucleus and subsequent transactivation of the human CTSK promotor. 16 Overexpression of MKK6, which enhanced p38 activity also stimulated CTSK gene expression and promotor activity. This phosphorylation of NFAT2 contrasts with the classical paradigm whereby calcineurin dephosphorylates both NFAT1 and NFAT2, leading to nuclear translocation and subsequent promotor activation of a spectrum of genes. 17,18 However, it is possible that both dephosphorylation and phosphorylation of different moieties of NFAT2 may induce translocation and subsequent transactivation of transcription. We have shown that RANKL treatment induces NFAT2 mrna expression in pre-osteoclasts. 19 Cyclosporine, which inhibits the phosphatase activity of calcineurin, thereby inhibiting NFAT activation, suppresses the stimulation of cathepsin K mrna expression by RANKL. 14 Furthermore, inhibitors of calcium signaling, such as BAPTA-AM and the calcium ionophore, A23187, reduce RANKL-induced cathepsin K mrna expression, NFAT2 overexpression markedly stimulates the rat cathepsin K promotor, and NFAT2 binds to a region of the rat cathepsin K promotor within 100 bp of the transcription initiation site. 14 Transfection of pre-osteoclasts with sirna specific to NFAT2 markedly reduces the induction of cathepsin K mrna by RANKL (Troen et al., unpublished results). RANKL treatment of cells also induces phosphorylation of the micropthalmia transcription factor (Mitf) via p38. 20 Mutations in the micropthalmia (mi) gene selectively affect osteoclast development and/or function and lead to osteopetrosis. 21,22 Mitf is the mi gene product and is a member of the helixloop-helix (HLH) leucine zipper family and directly regulates cathepsin K gene transcription. Dominant negative mutations of Mitf exhibit osteopetrosis and lack cathepsin K mrna and protein. 22 Overexpression of wild-type Mitf or TFE3, a member of the same transcription factor family, in cultured osteoclasts significantly enhanced cathepsin K expression. Mitf binds to three E-box motifs in the human CTSK promotor, of which mutation of any one significantly impairs stimulation of promotor activity by Mitf or TFE3. 22 In addition, both Mitf and PU.1 synergistically potentiated NFAT2 stimulation of human CTSK promotor activity. 16 Interestingly, osteoclasts that are null for both p21 and p27 fail to exhibit the marked stimulation of cathepsin K mrna

168 ANNALS NEW YORK ACADEMY OF SCIENCES by RANKL. 23 Since p21 and p27 are cyclin-dependent kinase inhibitors that appear to modulate the activity of HLH transcription factors, the authors hypothesize that p21 and p27 regulate cathepsin K expression via Mitf. Additional agents active in bone physiology stimulate cathepsin K expression. Retinoic acid can increase transcription of cathepsin K in isolated mature rabbit osteoclasts. 24 Extracellular matrix proteins that bind to osteoclast integrins, such as collagen type I, fibronectin, vitronectin, and osteopontin increased cathepsin K mrna expression. 25 Carbaprostacyclin, which is a ligand for peroxisome proliferator-activated receptor delta/beta (PPAR / ), induced cathepsin K mrna expression in mature rabbit osteoclasts concomitantly with bone resorption. 26 Antisense oligonucleotide to the PPAR / blocked the response to carbaprostacyclin. Intermittent mechanical stretching of mature osteoclasts stimulated cathepsin K mrna expression along with bone resorption. 27 Physiological inhibitors of osteoclast differentiation and activation can also directly suppress cathepsin K expression. Osteoprotegerin (OPG) inhibited cathepsin K expression in purified rabbit osteoclasts. 28 Estrogen inhibited bone resorption by isolated rabbit osteoclasts and also downregulated cathepsin K mrna. 29 In calvarial culture, estradiol suppressed cathepsin K mrna expression and cathepsin K activity in response to PTH, IL-1, IL-6, TNF-. 30 Calvarial cultures from ovariectomized mice exhibited enhanced synthesis of cathepsin K, likely due to an indirect effect mediated by increased expression of RANKL by osteoblasts. Interferon- (INF- ) inhibits cathepsin K mrna and protein expression in murine osteoclasts, while IL-1 stimulated cathepsin K expression. 31 These same investigators were unable to observe a significant effect of either TNF- or IL-6 on cathepsin K expression. We have also demonstrated that INF- also inhibits the stimulation of cathepsin K mrna and protein expression by RANKL in the preosteoclast RAW 264.7 cell line. 13 Sodium butyrate and trichostain, which are both histone deacetylase inhibitors, reduced the stimulation of cathepsin K by RANKL. 32 Members of the AP-1 family of transcription factors also regulate cathepsin K transcription. A mouse homologue of Jun dimerization protein 2 activates the cathepsin K promotor in transient transfections of mouse preosteoclast RAW cells. 33 Overexpression of junb stimulates cathepsin K promotor activity and also potentiates the stimulation by NFAT2. 14 CONCLUSION Therefore, activators and inhibitors of osteoclast bone resorption act, at least in part, through the stimulation and/or inhibition of cathepsin K gene expression. Some of these known and potential regulators of cathepsin expression are depicted in FIGURE 1. RANKL, NFAT, Mitf, and various components of AP-1 enhance osteoclast formation and bone resorption, whereas IFN-, calcitonin,

TROEN: REGULATION OF CATHEPSIN K GENE EXPRESSION 169 FIGURE 1. Regulation of cathepsin K expression. The osteoclast adheres tightly to bone, in part via binding of the v 3 integrin to the extracellular matrix proteins. The extracellular compartment is acidified by the H + -ATPase proton pump that actively transports H + ions produced by the action of carbonic anhydrase (CAII) in the cell. Chloride channels (CIC-7) maintain electrical balance within the cell by extruding Cl ions into the resorption lacuna. Cathepsin K and other matrix metalloproteineases (MMPs) are transported from the endoplasmic reticulum within lysosomes and other secretory vesicles and then released into the resorption lacuna. Agents that stimulate the osteoclast to produce increased amounts of cathepsin K include RANKL, NFAT, Mitf, TNF, PU.1, AP-1, TFE3 (a member of the microphthalmia transcription factor family), retinoic acid (RA), IL-1, peroxisome proliferator-activator receptor / (PPAR / ), stretching, and extracellular matrix proteins (ECM). Inhibitors of cathepsin K expression include estrogen, interferon- (IFN- ), osteoprotegerin, trichostatin A (TSA), sodium butyrate (NaB). (Adapted from reference 1, with permission.) estradiol, and calcium inhibit it. These agents appear to act correspondingly to alter cathepsin K gene and protein expression in order to stimulate and suppress the osteoclast s resorbing potential. However, it is not known whether the regulation of cathepsin K gene expression is inextricably linked with osteoclast formation and activation. It would be intriguing to investigate the possibility of genetically enhancing or reducing cathepsin K gene transcription without facilitating or impeding other osteoclast functions. Furthermore, while multiple RANKL-associated signaling cascades and nuclear-binding factors play a role in cathepsin K gene expression, additional studies are needed to determine whether specific pathways and transcriptional stimulators preferentially act

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