Title: Cathepsin K Activity-dependent Regulation of Osteoclast Actin Ring Formation and Bone Resorption
Abstract: Cathepsin K is responsible for the degradation of type I collagen in osteoclast-mediated bone resorption. Collagen fragments are known to be biologically active in a number of cell types. Here, we investigate their potential to regulate osteoclast activity. Mature murine osteoclasts were seeded on type I collagen for actin ring assays or dentine discs for resorption assays. Cells were treated with cathepsins K-, L-, or MMP-1-predigested type I collagen or soluble bone fragments for 24 h. The presence of actin rings was determined fluorescently by staining for actin. We found that the percentage of osteoclasts displaying actin rings and the area of resorbed dentine decreased significantly on addition of cathepsin K-digested type I collagen or bone fragments, but not with cathepsin L or MMP-1 digests. Counterintuitively, actin ring formation was found to decrease in the presence of the cysteine proteinase inhibitor LHVS and in cathepsin K-deficient osteoclasts. However, cathepsin L deficiency or the general MMP inhibitor GM6001 had no effect on the presence of actin rings. Predigestion of the collagen matrix with cathepsin K, but not by cathepsin L or MMP-1 resulted in an increased actin ring presence in cathepsin K-deficient osteoclasts. These studies suggest that cathepsin K interaction with type I collagen is required for 1) the release of cryptic Arg-Gly-Asp motifs during the initial attachment of osteoclasts and 2) termination of resorption via the creation of autocrine signals originating from type I collagen degradation. Cathepsin K is responsible for the degradation of type I collagen in osteoclast-mediated bone resorption. Collagen fragments are known to be biologically active in a number of cell types. Here, we investigate their potential to regulate osteoclast activity. Mature murine osteoclasts were seeded on type I collagen for actin ring assays or dentine discs for resorption assays. Cells were treated with cathepsins K-, L-, or MMP-1-predigested type I collagen or soluble bone fragments for 24 h. The presence of actin rings was determined fluorescently by staining for actin. We found that the percentage of osteoclasts displaying actin rings and the area of resorbed dentine decreased significantly on addition of cathepsin K-digested type I collagen or bone fragments, but not with cathepsin L or MMP-1 digests. Counterintuitively, actin ring formation was found to decrease in the presence of the cysteine proteinase inhibitor LHVS and in cathepsin K-deficient osteoclasts. However, cathepsin L deficiency or the general MMP inhibitor GM6001 had no effect on the presence of actin rings. Predigestion of the collagen matrix with cathepsin K, but not by cathepsin L or MMP-1 resulted in an increased actin ring presence in cathepsin K-deficient osteoclasts. These studies suggest that cathepsin K interaction with type I collagen is required for 1) the release of cryptic Arg-Gly-Asp motifs during the initial attachment of osteoclasts and 2) termination of resorption via the creation of autocrine signals originating from type I collagen degradation. Osteoclasts are monocyte-macrophage lineage-derived, large multinucleated cells. They are the major bone resorbing cells, essential for bone turnover and development. Active osteoclasts display characteristic membranes, including the ruffled border, attachment zone, and the basolateral secretory membrane. After attachment to bone, the ruffled border secretes enzymes and protons enabling the solubilization and digestion of the bone matrix. Osteoclasts express many proteases including cathepsins and matrix metalloproteases (MMPs) 2The abbreviations used are: MMP, matrix metalloprotease; catK, cathepsin K; catL, cathepsin L; TRAP, tartrate-resistant acid phosphatase; AR, actin ring; PBS, phosphate-buffered saline; wt, wild type. (for review see Refs. 1Goto T. Yamaza T. Tanaka T. J. Electron. Microsc. (Tokyo).. 2003; 52: 551-558Google Scholar, 2Delaisse J.M. Engsig M.T. Everts V. del Carmen Ovejero M. Ferreras M. Lund L. Vu T.H. Werb Z. Winding B. Lochter A. Karsdal M.A. Troen T. Kirkegaard T. Lenhard T. Heegaard A.M. Neff L. Baron R. Foged N.T. Clin. Chim. Acta.. 2000; 291: 223-234Google Scholar, 3Delaisse J.M. Andersen T.L. Engsig M.T. Henriksen K. Troen T. Blavier L. Microsc. Res. Tech.. 2003; 61: 504-513Google Scholar). However, it is the general consensus that cathepsin K (catK) is the major bone-degrading enzyme (4Inaoka T. Bilbe G. Ishibashi O. Tezuka K.I. Kumegawa M. Kokubo T. Biochem. Biophys. Res. Commun.. 1995; 206: 89-96Google Scholar, 5Brömme D. Okamoto K. Wang B.B. Biroc S. J. Biol. Chem.. 1996; 271: 2126-2132Google Scholar, 6Inui T. Ishibashi O. Inaoka T. Origane Y. Kumegawa M. Kokubo T. Yamamura T. J. Biol. Chem.. 1997; 272: 8109-8112Google Scholar, 7Saftig P. Wehmeyer O. Hunziker E. Jones S. Boyde A. Rommerskirch W. von Figura K. Proc. Natl. Acad. Sci. U. S. A.. 1998; 95: 13453-13458Google Scholar). Rapid cytoskeletal reorganization is essential for osteoclast function and formation of the specialized membranes. Bone resorption occurs within the sealing zone, which is formed by an actin ring structure. This can be identified as a solid circular belt like formation and consists of an actin filament core surrounded by actin-binding proteins such as talin, α-actinin, and vinculin, which link matrix-recognizing integrins to the cytoskeleton (8Marchisio P.C. Cirillo D. Naldini L. Primavera M.V. Teti A. Zambonin-Zallone A. J. Cell Biol.. 1984; 99: 1696-1705Google Scholar). The ruffled border is contained within this structure. The actin ring is initiated by the formation of podosomes, which represent dot-like actin structures of small F-actin containing columns surrounded by proteins also found in focal adhesion such as vinculin and paxillin (9Pfaff M. Jurdic P. J. Cell Sci.. 2001; 114: 2775-2786Google Scholar). It was previously thought that the sealing zone was formed by the fusion of podosomes after the osteoclast becomes activated (10Lakkakorpi P.T. Vaananen H.K. J. Bone Miner. Res.. 1991; 8: 817-826Google Scholar, 11Lakkakorpi P.T. Vaananen H.K. Microsc. Res. Tech.. 1996; 33: 171-181Google Scholar), but it has since been demonstrated that podosomes and the sealing zone are distinct structures (12Jurdic P. Saltel F. Chabadel A. Destaing O. Eur. J. Cell Biol.. 2006; 85: 195-202Google Scholar, 13Saltel F. Destaing O. Bard F. Eichert D. Jurdic P. Mol. Biol. Cell.. 2004; 15: 5231-5241Google Scholar). It should be noted that bone resorption only occurs when the sealing zone is formed and the actin ring is present (14Takahashi N. Ejiri S. Yanagisawa S. Ozawa H. Odontology.. 2007; 95: 1-9Google Scholar). Osteoclasts bind and interact with the bone surface through specific integrin receptors. The most abundant integrin present in osteoclasts is the αvß3 receptor also known as the vitronectin receptor (15Davies J. Warwick J. Totty N. Philp R. Helfrich M. Horton M. J. Cell Biol.. 1989; 109: 1817-1826Google Scholar, 16Nesbitt S. Nesbit A. Helfrich M. Horton M. J. Biol. Chem.. 1993; 268: 16737-16745Google Scholar). This receptor attaches to RGD sequence containing components of the bone matrix, e.g. vitronectin, osteopontin, and type I collagen (17Helfrich M.H. Nesbitt S.A. Lakkakorpi P.T. Barnes M.J. Bodary S.C. Shankar G. Mason W.T. Mendrick D.L. Vaananen H.K. Horton M.A. Bone.. 1996; 19: 317-328Google Scholar, 18Flores M.E. Norgard M. Heinegard D. Reinholt F.P. Andersson G. Exp. Cell Res.. 1992; 201: 526-530Google Scholar, 19Flores M.E. Heinegard D. Reinholt F.P. Andersson G. Exp. Cell Res.. 1996; 227: 40-46Google Scholar). This interaction enables the formation and regulation of the actin ring and therefore osteoclast activity (20Hynes R.O. George E.L. Georges E.N. Guan J.L. Rayburn H. Yang J.T. Cold Spring Harb. Symp. Quant. Biol.. 1992; 57: 249-258Google Scholar, 21Chellaiah M.A. Eur. J. Cell Biol.. 2006; 85: 311-317Google Scholar, 22van der Pluijm G. Mouthaan H. Baas C. de Groot H. Papapoulos S. Lowik C. J. Bone Miner. Res.. 1994; 9: 1021-1028Google Scholar). It has previously been shown that soluble RGD containing peptides added to cell supernatant are capable of inhibiting osteoclast binding and bone resorption (18Flores M.E. Norgard M. Heinegard D. Reinholt F.P. Andersson G. Exp. Cell Res.. 1992; 201: 526-530Google Scholar, 22van der Pluijm G. Mouthaan H. Baas C. de Groot H. Papapoulos S. Lowik C. J. Bone Miner. Res.. 1994; 9: 1021-1028Google Scholar, 23Horton M.A. Taylor M.L. Arnett T.R. Helfrich M.H. Exp. Cell Res.. 1991; 195: 368-375Google Scholar, 24King K.L. D'Anza J.J. Bodary S. Pitti R. Siegel M. Lazarus R.A. Dennis M.S. Hammonds Jr., R.G. Kukreja S.C. J. Bone Miner. Res.. 1994; 9: 381-387Google Scholar). This study investigates the effect of collagen degradation fragments on osteoclast activity. Soluble type I collagen and the bone powder of murine long bones were subjected to digestion reactions by the cysteine proteases, catK and catL, and the interstitial collagenase, MMP-1. The effect of these degradation products on osteoclasts was investigated by monitoring actin ring and resorption pit formation. We further investigated the role of cathepsins using catK- and catL-deficient mice. Finally, we looked in more detail at the effect of collagen, as a cell adhesion matrix, on osteoclast activity. Mouse Models—In these studies, the mouse models used were WT C57BL/6 (The Jackson Laboratories), catK knock-out mice 129:C57BL/6 (7Saftig P. Wehmeyer O. Hunziker E. Jones S. Boyde A. Rommerskirch W. von Figura K. Proc. Natl. Acad. Sci. U. S. A.. 1998; 95: 13453-13458Google Scholar), and catL knock-out mice 129:C57BL/6 (25Roth W. Deussing J. Botchkarev V.A. Pauly-Evers M. Saftig P. Hafner A. Schmidt P. Schmahl W. Scherer J. Anton-Lamprecht I. Von Figura K. Paus R. Peters C. Faseb. J.. 2000; 14: 2075-2086Google Scholar). Osteoclast Isolation from Neonatal Murine Long Bones—Mature osteoclasts were isolated from 6-day-old mouse long bones (26Hoebertz A. Arnett T.R. Helfrich M.H. Ralston S.H. Bone Research Protocols. 80. Humana Press, Totowa, NJ2003: 53-64Google Scholar). Long bones were isolated and collected in α-modified minimal essential medium (α-MEM, Invitrogen) supplemented with 10% fetal bovine serum, 2 mm l-glutamate (Invitrogen), they were then diced into small pieces, and bone cells were released by gentle pipetting. The resulting cell suspension (without bone pieces) was then plated onto collagen-coated slides in a 24-well plate and incubated at 37 °C (95% air and 5% CO2). After 2 h, nonattached cells were washed away, and cells were treated with 10 μg/ml lipopolysaccharide (Sigma-Aldrich) and additional treatment as specified below. Cells were then cultured at 37 °C (5% CO2) for 24 h. To confirm the presence of osteoclasts, cells were stained for tartrate-resistant acid phosphatase activity (TRAP), an osteoclast marker, according to the manufacturers' instructions (Sigma-Aldrich). The number of positive TRAP-stained cells was about 50% of the total number of cells in preparations from wild type, catL-/- and catK-/- cells. Cell Treatments—GRGDS and SDGRG (Sigma-Aldrich) were used at a final concentration of 100 μg/ml. For repeat RGD dose experiments, the media was changed every 2 h for 8 h to fresh media containing GRGDS (5-50 μg/ml). The irreversible (potent, non-selective) vinyl sulfone cathepsin inhibitor, LHVS, (27Palmer J.T. Rasnick D. Klaus J.L. Brömme D. J. Med. Chem.. 1995; 38: 3193-3196Google Scholar, 28Riese R.J. Wolf P. Bromme D. Natkin L.R. Villadangos J.A. Ploegh H.L. Chapman H.A. Immunity.. 1996; 4: 357-366Google Scholar) was used at a final concentration of 5 μm. The broad spectrum MMP inhibitor GM6001 (Chemicon, Temecula, CA) was used at a final concentration of 5 μm. All cell treatments were for 24 h unless otherwise stated. Proteases—Recombinant human cathepsins K and L were expressed using the Pichia pastoris expression system (29Linnevers C.J. McGrath M.E. Armstrong R. Mistry F.R. Barnes M. Klaus J.L. Palmer J.T. Katz B.A. Brömme D. Protein Sci.. 1997; 6: 919-921Google Scholar, 30Bromme D. Nallaseth F.S. Turk B. Methods.. 2004; 32: 199-206Google Scholar). Recombinant human MMP-1 was a generous gift from Dr. Chris Overall (Centre for Blood Research, University of British Columbia, Canada). Fluorescent Staining, Actin Ring Formation of Osteoclasts—Cells were washed with PBS, fixed with 3.7% formaldehyde in PBS, and permeabilized with 0.2% Triton X-100 for 10 min. Osteoclast actin rings were visualized using FITC-phalloidin (Sigma-Aldrich) (1:50 dilution) staining as previously described (31Graebert K.S. Bauch H. Neumuller W. Brix K. Herzog V. Exp. Cell Res.. 1997; 231: 214-225Google Scholar). After staining, cells were washed with PBS, and the nuclei were stained with bisbenzimide (Sigma-Aldrich) (2 μg/ml) for 5 min and then rinsed with water. Cells were mounted with Fluoromount (Sigma-Aldrich). Actin rings were visualized fluorescently using a Leica DMI 6000B microscope (Leica Microsystems, Inc, Richmond Hill, ON) and the total number per slide was counted. Osteoclasts were identified by the presence of at least 2 nuclei. Typically osteoclasts contained between 3-7 nuclei, no distinction was made between large and small osteoclasts. If an osteoclast displayed one or more actin rings it was denoted as actin ring positive (AR+), osteoclasts without or disrupted actin rings were actin ring negative (AR-). The ratio of normal versus disrupted actin rings was calculated. An actin ring was considered disrupted if less than half of it exhibited typical actin ring morphology (10Lakkakorpi P.T. Vaananen H.K. J. Bone Miner. Res.. 1991; 8: 817-826Google Scholar). Osteoclasts were also visualized by confocal microscopy using a Nikon confocal C1 microscope with EZ-C1 software (Nikon Instruments Inc, Mississauga, ON). Resorption Assay—Cells were plated out onto dentine discs (Osteosite Dentine Discs, Immunodiagnostic Systems Inc, Fountain Hills, AR) in 96-well plates as previously described (26Hoebertz A. Arnett T.R. Helfrich M.H. Ralston S.H. Bone Research Protocols. 80. Humana Press, Totowa, NJ2003: 53-64Google Scholar). After 2 h, discs were removed and placed in 6-well plates containing media (α-MEM at around pH 7.0) and test substance. There were typically 4 dentine discs per group. After 24 h, slices then stained for TRAP, which allowed the identification of osteoclasts (TRAP+ with over 2 nuclei). Once osteoclasts were counted, cells were removed with 5% sodium hypochlorite for 10 min. Discs were rinsed with water and stained with 1% (w/v) toluidine blue in 0.5% sodium borate for 30 s and then washed with water. The number and the area of resorption pits were then measured by light microscopy using Openlab 4.0.3 software. Results are expressed as the number of resorption pits, and total area resorbed per dentine disc. Collagen Digests—Soluble type I collagen (5 mg/ml calf skin) (USB, Cleveland, OH) was incubated with human catK (200 nm) or human catL (200 nm) in sodium acetate buffer, pH 5.5, containing 2.5 mm dithiothreitol and EDTA or with p-aminophenylmercuric acetate (APMA) activated MMP-1 (50 nm) in 5 mm CaCl2, 50 mm HEPES, 100 mm NaCl, pH 7.2. Total volume of each reaction was 100 μl. Collagen digestions were performed at 28 °C in the absence and presence of 400 nm chondroitin 4-sulfate (Sigma-Aldrich) for 8 h (chondroitin 4-sulfate concentration based on an average molecular mass of 30 kDa). In both collagen and bone digests enzymes were inactivated by raising the pH of the reaction to 7.2 and then heating at 50 °C for 2 min. In cell treatments where collagen degradation products were added to media, 20 μl of pH-adjusted collagen digest was added to 1 ml of media. CatK-digested collagen was also subject to degradation by trypsin (Sigma-Aldrich), the pH was altered to 7.6 with a Tris buffer (100 mm final concentration) trypsin was added at 1 μm for 4 h at 30 °C. Bone Digests—Long bones from 4-6-week-old wild-type mice were isolated, cleaned, and the epiphysis and bone marrow removed. Lipids were removed by incubating long bones overnight in xylene, bones were then frozen and crushed with a pestle and mortar to obtain bone powder. Bone powder was washed three times with sodium acetate buffer before enzyme was added. Human catK or catL were added to bone powder (15 mg with 100-μl reaction volume) at a concentration of 400 nm and incubated at 28 °C for 24 h. Samples were then spun down, the soluble fraction was removed and after its pH was neutralized 20 μl was added to 1 ml of media for cell treatments. Collagen-coated Coverslips—Thin collagen coatings were generated as described by R&D systems (R&D systems, Inc. Minneapolis, MN) on glass coverslips. Soluble type I collagen (final concentration 50 μg/ml) was diluted in 500 μl of 0.02 mm acetic acid and 200 μl added per coverslip. Coverslips were incubated at room temperature for 1 h, residual volume was removed, and coverslips were washed with PBS before cells were added. Type I collagen was also pre-degraded with 200 nm catL or 200 nm catK in 100 mm sodium acetate buffer, pH 5.5 at 28 °C. Digested collagen was then added at the same concentration as undigested collagen to coverslips. Gel Electrophoresis—Collagen degradation was analyzed by SDS-PAGE. Samples (1.5 μg per well) were boiled for 5 min with 2× reducing SDS-PAGE sample buffer and separated using 4-20% gradient gels (1.5 h at 125 V) (Invitrogen), 5 μl of prestained protein ladder (PAGE, Invitrogen) was included for orientation. Bands were visualized using Coomassie Brilliant Blue R 250 (0.5 mg/l, in 40% methanol and 10% acetic acid) and were then destained (40% methanol, 10% acetic acid). Statistical Analysis—Experiments were performed in duplicate three times using osteoclast cultures from 3 different mice. Data are expressed as mean ± S.D. The statistical significance of the difference between the control and the experimental group was determined by Student's t test. In bone resorption assays, experiments were performed four times and comparisons between control and each treatment group was made using the Mann Whitney U test. Effects were considered statistically significant when p ≤ 0.05. Effect of Proteolytically Degraded Soluble Collagens and Bone Powder on Actin Ring Presence in Wild-type Osteoclasts—This study investigated the effect of collagen fragments on osteoclast actin ring presence using neonatal murine osteoclasts seeded onto type I collagen. As mature osteoclasts were used in experiments a differentiation stage was not required. Type I collagen was used as a matrix as it is a well-defined catK substrate, and this substrate also decreases the matrix variability often found with bone slices. After attachment to collagen slides the osteoclasts were incubated for 24 h before the presence of actin rings was investigated. Under our conditions, 24-h incubations resulted in the highest incidence of actin rings and so this time point was used for all subsequent experiments. Wild-type osteoclasts were first treated with a synthetic peptide containing the RGD sequence. The majority of untreated osteoclasts revealed well-formed actin rings (Fig. 1A), whereas, as expected, cells treated with GRGDS peptide displayed mainly disrupted actin rings (Fig. 1B). The quantification of intact and disrupted actin rings in wild-type osteoclasts is shown in Fig. 1C. The addition of 100 μg/ml GRGDS peptide to wild type osteoclasts resulted in a 50% decrease in actin ring formation (Fig. 1C); in contrast the reverse sequence peptide SDGRG showed no effect on actin rings as it does not affect integrin function. Similar effects on osteoclast activity have previously been described for RGD containing proteins and peptides (32Masarachia P. Yamamoto M. Leu C.T. Rodan G. Duong L. Endocrinology.. 1998; 139: 1401-1410Google Scholar, 33Nakamura I. Tanaka H. Rodan G.A. Duong L.T. Endocrinology.. 1998; 139: 5182-5193Google Scholar, 34Nakamura I. Pilkington M.F. Lakkakorpi P.T. Lipfert L. Sims S.M. Dixon S.J. Rodan G.A. Duong L.T. J. Cell Sci.. 1999; 112: 3985-3993Google Scholar). We found that the GRGDS peptide had an accumulative inhibitory effect over time. A low concentration of RGD (10-50 μg/ml) left in contact with cells over 8 h had minimal effect on osteoclast actin rings. However when the same low doses were repeatedly administered to the cells every 2 h accompanied by a media exchange, the percentage of actin rings present was strongly reduced (Fig. 1D). Next, we subjected triple-helical type I collagen which cumulatively contains 7 cryptic RGD motifs to degradation by catK, catL, and MMP-1 in the presence of chondroitin sulfate to mimic the presence of glycosaminoglycans in bone. We have previously demonstrated that glycosaminoglycans specifically modulate the collagenolytic activities of cathepsins (35Li Z. Yasuda Y. Li W. Bogyo M. Katz N. Gordon R.E. Fields G.B. Bromme D. J. Biol. Chem.. 2004; 279: 5470-5479Google Scholar). As described, CatK digestion resulted in a complete degradation of type I collagen, whereas the catL-mediated digest was minimal and the MMP-1 mediated digest revealed typical 3/4 and 1/4 fragments (35Li Z. Yasuda Y. Li W. Bogyo M. Katz N. Gordon R.E. Fields G.B. Bromme D. J. Biol. Chem.. 2004; 279: 5470-5479Google Scholar, 36Li Z. Hou W.S. Bromme D. Biochemistry.. 2000; 39: 529-536Google Scholar) (Fig. 2A). F-actin staining of murine wild-type osteoclasts treated with catK-degraded type I collagen (final concentration 100 ng/ml) for 24 h showed a 75-80% inhibition of actin ring formation in wild-type cells (Fig. 2). No statistically significant inhibition was found after treatments with catL or MMP-1-digested collagen although catL-predigested type I collagen revealed a trend toward actin ring inhibition (Fig. 2A). There was also no effect on the percentage of osteoclasts with actin rings when undegraded collagen or vehicle (sodium acetate buffer) alone was added (results not shown). The inhibitory effect of catK-digested collagen was lost when it was subjected to further degradation by trypsin (Fig. 2C), known to cleave after arginine or lysine residues and thus likely to destroy the RGD moiety (37Perona J.J. Craik C.S. Protein Sci.. 1995; 4: 337-360Google Scholar). Prior to the addition of the tryptic digest to the cells, trypsin was heat-inactivated. We also incubated the osteoclasts in the presence of the soluble products of protease-pretreated murine bone powder. Similar to the observation made with predigested soluble collagen, catK-digested bone powder revealed the strongest actin ring reduction. Up to 95% of the cells displayed disrupted actin ring structures suggesting a complete inhibition of the osteoclast activity (Fig. 2A). In contrast no effect was observed when cells were exposed to bone powder pretreated with catL. These results were also reflected in bone resorption assays performed on dentine discs. Both the number of resorption pits and the area resorbed per disc decreased on addition of catK-digested bone powder with no change in osteoclast number (Fig. 2B). As expected, catL-pretreated bone powder had no effect on bone resorption. Actin formations observed in osteoclasts treated with catK degraded collagen showed podosome-like structures, often with actin forming clumps in a similar manner to cells treated with GRGDS (see Fig. 1B). Confocal microscopy showed that osteoclasts treated with catK-digested collagen fragments also displayed signs of cell retraction (Fig. 2D) similar to that observed when osteoclasts are treated with GRGDS peptide (23Horton M.A. Taylor M.L. Arnett T.R. Helfrich M.H. Exp. Cell Res.. 1991; 195: 368-375Google Scholar). The observed reduction in actin ring numbers was not thought to be due to a decrease in attached cells as all experiments had comparable osteoclast numbers. It has been previously shown that actin ring formation can be disrupted with no effect on osteoclast differentiation, survival, and attachment (38Nakagawa H. Takami M. Udagawa N. Sawae Y. Suda K. Sasaki T. Takahashi N. Wachi M. Nagai K. Woo J.T. Bone.. 2003; 33: 443-455Google Scholar). Effect of Cysteine and Metalloprotease Inhibitors on Actin Ring Formation in Wild-type Osteoclasts—After demonstrating that catK-predigested RGD peptide containing substrates affect the formation of actin rings, we analyzed the effect of protease inhibition in osteoclasts. Considering that cathepsin K activity generates active RGD peptides, we expected that the inhibition of cathepsin K and thus eliminating the generation of soluble RGD fragments would stabilize or increase the presence of actin rings. However, the addition of 5 μm of the cell-permeable broad spectrum cathepsin inhibitor, LHVS, to wild-type osteoclasts resulted in a 50% reduction of actin ring formation when compared with untreated osteoclasts (Fig. 3A). This suggests that cathepsin activities are required for the initial formation and/or maintenance of actin rings. At 5 μm concentration, LHVS is a potent inhibitor of cathepsins K, L, and S (27Palmer J.T. Rasnick D. Klaus J.L. Brömme D. J. Med. Chem.. 1995; 38: 3193-3196Google Scholar). In contrast, the broad spectrum metalloprotease inhibitor GM6001 had only a weak and statistically non-significant effect on the reduction of actin rings. Effect of catK and -L Deficiency on Actin Ring Formation—As the broad spectrum cathepsin inhibitor, LHVS, was able to decrease actin ring formation, we next aimed to identify the individual cathepsin responsible for this inhibition by analyzing catK- and catL-deficient osteoclasts. CatK-deficient cells showed a similar suppression of actin rings as wild-type osteoclasts treated with LHVS. Both conditions displayed less than 50% of actin rings compared with untreated wild-type osteoclasts. The addition of catK-predigested soluble type I collagen and bone powder to catK-deficient osteoclasts further suppressed the percentage of cells exhibiting intact actin rings (Fig. 4A). On the other hand and similar to wild type osteoclasts, catL-pretreated collagen or bone powder did not affect the number of actin rings (Fig. 4A). On the contrary, catL-deficient osteoclasts were statistically undistinguishable from wild-type cells in the absence or presence of collagen/bone digest mixtures (Figs. 4B and 2A) suggesting that the loss of catL activity is not critical to osteoclast activity. Treatment of cathepsin-deficient cells with LHVS had no effect on catK-deficient cells whereas catL-deficient cells revealed a strong reduction in intact actin rings (60%) similar to that observed in wild-type cells in the presence of LHVS (Fig. 3) and of catK-deficient cells in the presence and absence of the cysteine protease inhibitor (Fig. 4C). In each case the percentage of osteoclasts displaying actin rings was reduced to between 15 and 30% indicating that catK alone is critically involved in actin ring formation. On the other hand, the metalloprotease inhibitor GM6001 had no significant effect on both cathepsin-deficient cell types (Fig. 4C). Effect of catK-predigested Type I Collagen Matrix on CatK-/- Osteoclasts and LHVS-treated Wild-type Osteoclasts—Both the addition of extracellular RGD-containing peptides or proteolytic fragments and the inhibition of catK inhibit actin ring formation and thus indicate the involvement of cathepsins, in particular catK, in the generation as well as the dissolution of actin rings following at least two pathways. To investigate whether cathepsin activities are required for the initial exposure of cryptic integrin binding sites in the collagen matrix and subsequently allowing actin ring formation, we predigested type I collagen with catK or catL and seeded catK-deficient osteoclasts on the predigested substrate matrix. CatK-deficient osteoclasts revealed a significant increase in actin ring numbers when grown on catK-predigested but not on catL-predigested collagen matrix (Fig. 5A). In contrast, there was no difference observed for wild-type cells on untreated, catK-, or catL-predigested matrices. However, similar to catK-deficient cells, LHVS treated wild-type cells seeded on catK-predigested matrix increased the actin ring content compared with wild-type cells treated with LHVS seeded on intact matrix (Fig. 5B). Although there was an increase in the number of actin rings, osteoclasts did not reach the levels of actin rings observed when untreated osteoclasts are seeded on intact collagen. These results suggest that catK activity is required to expose cryptic RGD motifs in type I collagen and to form actin rings. The regulation of individual osteoclast activity remains unclear. This study aimed to investigate whether catK, responsible for bone degradation and highly expressed in osteoclasts, is capable of directly regulating osteoclast action by its extracellular matrix-degrading activity. To do this we exploited a cell based actin ring assay and minimized the effects of the extracellular matrix by using a single component (collagen type I) system representing the major and biologically relevant cathepsin K substrate (90% of the organic bone matrix is type I collagen). Mature murine osteoclasts were seeded on the collagen matrix and treated with catK, catL, or MMP1 predegraded as well as undegraded soluble type I collagen or bone powder and the percentage of osteoclasts displaying actin rings were determined. Only soluble type I collagen or type I collagen containing bone powder degraded by catK was capable of decreasing the percentage of active osteoclasts from wild-type mice (Fig. 1), which was in a similar range to the inhibition found after the addition of the synthetic control GRGDS peptide. Triple helical type I collagen which contains in its α1 and α2 chains a total of 7 RGD motifs, is cleaved by catK at multiple sites and generates low molecular weight fragments (39Garnero P. Borel O. Byrjalsen I. Ferreras M. Drake F.H. McQueney M.S. Foged