Characterization of Phagosomal Subpopulations Along Endocytic Routes in Osteoclasts and Macrophages1

J. Biochem. 130. 82:2-031,20

Characterization of Phagosomal Subpopulations along Endocytic Routes in Osteoclasts and Macrophages1

Eiko Sakai, Hisatsugu Miyamoto ,7 Kuniaki Okamoto, Yuzo Kato,* Kenji Yamamoto,$' and Hid eaki Sakai* Departments of Pharmacology and Orthodonties. Nagasaki University School of Dentistry; Nagasaki and *Department of Pharmacology, Kyushu University Graduate School of Dental Science. Fukuoka 812-8582

Received September 6, 2001, accepted October 4, 2001

Modifications occurring during the transformation of phagosomes into mature phagoly sosomes were investigated in osteoclast-like cells (OCLs) and macrophages using latex beads as markers for the isolation of phagosomal compartments (LBC) at different time points after phagocytosis. In OCLs, newly formed LBC acquired cathepsin K, tartarate resistant phosphatase (TRAP), lysosome-associated membrane protein-1 (Lamp-1) , and cathepsin D, and rapidly lost annexin II in a time-dependent manner . The levels of Rab7 and c-Src in OCLs initially increased and then gradually decreased during the transfor mation from early to late endosomal LBC or phagolysosomes . Receptor activator of NF- KB (RANKL) significantly increased the LBC levels of cathepsin K, TRAP , and c-Src, whereas calcitonin decreased the LBC levels of cathepsin K, TRAP, and Rab7, indicating that the transformation of early to late endosomal elements and lysosomes in OCLs is also regulated by osteoclastogenesis regulatory factors. On the other hand , changes in th e LBC levels of Lamp-1, cathepsin D, and annexin II in macrophages were comparable to those in OCLs. However, contrary to osteoclastic LBC, Rab7 levels of macrophage LBC decreased in a time-dependent manner. Macrophage LBC were devoid of cathepsin K. TRAP, and c-Src in all transformation stages. These observations suggest that OCLs and macrophages have different phagosome maturation mechanisms that involve the specific and regulated acquisition of markers from endocytic organelles. The results also demonstrate that the use of LBC is a useful system in which to identify and characterize molecules involved in these different endocytic pathways.

Key words: endocytosis, macrophage, osteoclast, phagosome.

Phagocytosis is the process by which cells internalize large may confer cell-specific properties on endosomal subpopul particulate materials destined to be degraded by an endo tions through the acquisition of marker molecules fro some/lysosome system. The more active phagocytic cells are characteristic endocytic organelles. However, it is poor mononuclear phagocytes (monocytes and macrophages) understood whether endocytic pathways vary in differe and polymorphonuclear leucocytes (neutrophils and eosino cell types. phils). Although this process is important in controlling in Osteoclasts are polarized and multinucleated cells r fectious diseases and in the turnover of senescent cells, sponsible for bone resorption. The most characteristic fe very little is known about the events occurring after inter lure of actively resorbing osteoclasts is the presence of nalization that lead to the transformation of newly formed ruffled border, a convoluted plasma membrane structu phagosomes into mature phagolysosomes. The available adjacent to the bone surface, and the clear zone. These cc evidence indicates that phagosomes successively fuse with dissolve both bone mineral and matrix proteins by acti different intracellular compartments including late endo secretion of protons and proteinases through the cuff somes and lysosomes and thereby acquire marker mole membrane into the extracellular resorption lacunae (1-( cules from these endocytic organelies. The fusion events be It is also known that bone degradation products are enc tween early phagosomes and intracellular compartments cytosed from the ruffled border membrane and transport and secreted to the opposite extracellular space via ti 1 This work -- supported in part by a Grant-in-Aid (09771541) basolateral membrane (7, 8)- This transcytotic pathway from the Ministry of Education, Science. Sports and Culture of .la- important in bone remodeling but poorly understood, Pre, pan. ously collagen fragment-containing intracellular vacuol 2 To whom correspondence should be addressed. Tel: 081-92-642- 6337, Fa+81-92-642-6342), E-mail: [email protected] have been observed in actively resorbing osteoclasts c Abbreviation. Lamp, lysosome-associated membrane protein; LBC, localizing with cathepin K (9) and cathepsin E (h latex bead-containing compartments: M-CSF, macrophage colony Cathepsin K is a specific cysteine proteinase that is excl stimulating factor; OCL, osteoclast-like cell. PAGE. polyacrylamide sively expressed in osteoclasts (3, 4, 11, 12), whereas cath gel electrophoresis; PBS, phosphate-buffered saline; RANKL, recep psin E is a specific aspartic proteinase that has be tor activator of NF-KB ligand, SDS, sodium dodecyl sulfate: TRAP identified in limited sources such as the gastrointestin tartarate-resistant acid phosphatase. tract and lymphoid tissues (13). Since both enzymes a (C) 2001 by The Japanese Biochemical Society, confined exclusively to the ruffled border membrane al

Vol. 130, No. 6, 2001 823 824 E. Sakai et al.

the eroded bone surface, it is most likely that they play a stroma cells were removed by treatment with 0.0011 pro role in the extracellular degradation of bone matrix pro nase E (Calbiochem, La Jolla, CA, USA) in 0.02% EDTA. teins. However, the co-localization of collagen fragments Further incubation was carried out in the presence of 20 with these enzymes in some endocytic vacuoles also sug ng/ml macrophage-colony stimulating factor (M-CSF) (R&D gests their involvement in the intracellular degradation of Systems, Minneapolis, MN, USA). More than 80% of the bone matrix proteins in the course of transcytosis. final adherent cells were TRAP-positive multinucleated Macrophages are also highly endocytic cells that are cells and mononuclear cells. J774 mouse macrophages were capable of internalizing a broad spectrum of both soluble obtained from Riken Cell Bank (Tokyo) and grown in 100 and particulate molecules. It is now known that the phago mm dishes in Dulbecco's modified Eagle's medium contain- cytotic system in macrophages is involved in immune pro ing 10% FBS, and 1% glutamine (21). cesses, in addition to the inactivation of bacterial endo Phagosome Formation and Cell Fractionation-The for toxins and the metabolism of lipids, steroids, and proteins. mation and isolation of phagosomes were carried out by the Although much progress has been made in elucidating the method of Desjardins et al. (15, 16) as described previously, properties of phagocytosis in macrophages, little is known with slight modifications. Phagosomes in OCLs and mac about the regulation of the transformation of early phago rophages were formed by the internalization of latex beads somes to mature phagolysosomes. (1.7 g.m diameter, 2.57 suspension, yellow, Fluoresbrite In the present study we have attempted to characterize TM, Polysciences, PA, USA) diluted 1:50 in culture medium phagosomal subpopulations isolated from both osteoclasts at 37•Ž for 60 min. The cells were then washed in phos and macrophages by the latex bead method. This method is phate-buffered saline (PBS) (3 x 10 min) on ice, followed by a useful technique for qualitative and quantitative bio- a chase period of up to 24 h in medium without beads at chemical analyses of head-associated proteins at different 37•Ž. The cells were disrupted in homogenization buffer [3 stages of phagosome formation and maturation (14-16). mM imidazole, pH 7.4, containing 8.55% (w/w) sucrose, 2 The results show that latex bead-containiiing compartments mM phenylmethylsulfonyl fluoride, 1 ƒÊg/ml chymostatin, 1

LBC %in both cell types gradually transform with time and g/ml E-64, 1 ƒÊg/ml leupeptin, 1 ƒÊg/ml pepstatin] by nitro ƒÊ transiently display a protein composition characteristic of gen cavitation for 20 min at 300 psi in a bomb (Pair Instru each cell type. ment, Moline, IL) at 4•Ž. After centrifugation at 1,500 rpm for 7 min to remove nuclei and unbroken cells, the superna

MATERIALS AND METHODS tants containing the latex bead-containing phagosomal compartments (LBC) were subjected to stepwise sucrose Antibodies and Chemicals-Specific antibodies against gradient centrifugation. The LBC supernatant was adjust- cathepsin B (17), cathepsin D (18), and the C-terminal pep- ed to 40% sucrose by adding 627 sucrose solution, and tide of cathepsin K (19) were prepared as described previ then loaded on a 1-ml cushion of 621, sucrose. Then, 2 ml of ously. Antiserum against TRAP was a kind gift from Dr. G. .357 sucrose, 2 nil of 251 sucrose, and 2 ml of 10% sucrose Andersson (Division of Immunology Microbiology, Pathol solution (pH 7.4) were layered. Centrifugation was carried ogy, and Infectious Diseases Karolinska Institute, Univer out at 24,000 rpm for 1 h in a SW40 Hitachi swinging rotor. sity Hospital. Sweden). Specific antibodies against Rab7 The LBC fractions were collected from the 10/251 sucrose and Rab5 were kind gifts from Dr. E. Kominami (Depart interface. The collected LBC fractions were washed with ment of Biochemistry Juntendo University, Tokyo). Anti- PBS. The purity of the final LBC fractions was evaluated bodies against Lamp-1 was a kind gift from Dr K. Akasaki by electron microscopy (Faculty of Pharmacy and Pharmaceutical Sciences, Fuku Electron Microscopy-Cultured OCLs and macrophages yama University, IIiroshinra). Antibodies against Rab3, c- were fixed with 2, paraformaldehyde, 1% glutaraldehyde, Src, and annexin II were purchased from Santa Cruz Bio- and 0.02% CaCl2 in 0.05 M cacodylate buffer (pH 7.4). technology (Santa Cruz, CA, USA). Recombinant human Post fixation was carried out in 1% OsO, in 0.05 M cacody RANKL (sRANKL) was purchased from Pepro Tech EC late buffer (pH 7.4), for 2 h, followed by dehydration in eth LTD )London, UK). Eel calcitonin (elcatonin) was a kind anol, and embedded in Quetol 812 resin. Ultra-thin sec gift from Asahi Chemical Industry (Tokyo). All chemicals tions were cut with a diamond knife and examined using a used were of analytical grade. Hitachi H-800 electron microscope. Culture of Osteoclast-Like Cells (OCLs) and .177.1 Mouse WesternBlot Analysis The kinetic association of various Macrophages-Bone marrow cells were obtained from the molecules in endocytic organelles with LBC was deter- tibiae and femora of 6-8-week-old male ddY mice. Bone mined by Western immunoblotting and densitometric scan marrow cells (5•~10`' cells) were co-cultured with primary- ning analysis. The same number of LBC, as determined by cultured osteoblasts (1 x 105 cells) prepared from the calva the fluorescence intensity of the latex beads at 480 nor (ex- riae of newborn ddY mice in ƒ¿-minimal essential medium citation at 380 nm), was used in each experiment. At each (,MEM) containing 10% fetal bovine serum (FBS), lƒ¿,25- time point, the LBC fractions from both cell types were dis dihydroxyvitamin D3 [1ƒ¿,25(OH)2D3;] (10" M, kindly pro- solved in SDS buffer (2%,SDS/100 mM dithiothreitol/10% vided from Teijin, Tokyo), and prostaglandin E2 110 " M) in glycerol/0.0025% bromophenol blue in 62.5 mM Tris-HCl, 100-mm-diameter dishes coated with collagen gel (Nitta pH 6.8), and then subjected to SDS-PAGE under reducing Gelatin, Osaka) as described previously (30). After 7 days of conditions. Proteins separated in the gels were transferred culture, the cells were isolated from the gels by treatment onto polyvinylidene difluoride membranes. The antigenic with 0.1% collagenase (type 3, Worthington Biochemical, sites were detected with an enhanced ECL kit (Amersham, NJ, USA) and replated on plastic dishes for 2 h at 37•Ž in a Buckinghamshire, UK). 5% CO, atmosphere. Weakly adherent bone marrow and

J. Biochem. Endocytic Transports in Osteoclasts and Macrophages 825

ization of these enzymes in osteoclasts was compared with RESULTS that in macrophages (J774). Macrophages were devoid of the immunoreactivity for cathepsin K and activity staining Microscopic View of Phagocytosing OCLs-OCLs derived for TRAP. However, the intracellular reactivity for cathep from co-cultures of primary osteoblasts and bone marrow sins D and B was more intense than in OCLs. These two cells were plated on plastic dishes for 2 h at 37•Ž . Weakly antigens were exclusively confined to the intracellular vesi adherent bone marrow and stroma cells were removed by cles and vacuoles throughout the cytoplasm. treatment with pronase E and EDTA. More than 80% of When the isolated OCLs were incubated with latex beads the cells in this preparation were positive for TRAP, a for 60 min at 37•Ž, most of the cells rapidly phagocytosed marker for osteoclasts (Fig. 1A). The isolated OCLs were the latex beads (Fig. 3, A and B). Within a 4 h chase, newly immunostained with specific antibodies against cathepsins formed LBC were localized in the center of the cytoplasm K, D, and B (Fig. 2). Preferential immunoreactivity for (Figs. 1B and 3, C and D). After a 24 h chase, LBC cathepsin K was observed at the cell surface. Cathepsin K migrated to a more central area of the cytoplasm (Fig. 3, E was also detected in some endocytic vacuoles in these cells. and F). At the electron microscopic level, LBC were seen to Cathepsin D immunoreactivity in the cells was found in be initially localized at or in the vicinity of the apical cell vesicles and vacuoles distributed throughout the cyto surface (Fig. 4A). After a 4 h of chase, LBC were sur- plasm. This antigen was not detected on the cell surface of rounded by electron dense endosome-like ornagelles near OCLs. Conversely, immunoreactivity for cathepsin B was barely detectable in OCLs. The immunohistochemical local-

Fig. 1. Morphological evaluation of the OCLs preparation from mouse bone marrow cells using activity staining for TRAP. Mouse bone marrow cells were co-cultured with mouse pri mary osteoblasts in the presence of la,25(OH),D, (10"' MI and PGE2 (100 MI for 7 days. After treatment with 0.1% collagenase, the de tached cells were inoculated and cultured for 2 h on dishes. After re Fig. 2. Morphological evaluation of OCLs and J774 macroph moval of the less-adherent bone marrow and stroma cells with ages, using various marker molecules for intracellular or pronase E/EDTA, the remaining adherent cells were stained for ganelles. Following fixation with 4% paraformaldehyde, both cell TRAP and methyl-green (A). The OCLs fraction was incubated with types were immunostained with antibodies specific for cathepsin K latex beads for 1 h to allow phagocytosis to occur. After internaliza (A, B), cathepsin D (E, F), and cathepsin B )G, H) or normal rabbit tion, the cells were washed and chased for 4 h. Following fixation IgG (I, J). Each cell type was also stained for TRAPase (C, D). Orig with 4% paraformaldehyde (PFA), the cells were stained for TRAP inal magnification •~100. TRAP-positive OCLs contain the phagocy (B). Original magnification •~25. tosed latex beads.

Vol. 130, No. 6, 2001 826 E. Sakai et al. the ruffling membrane (Fig. 4B) and inside the cells (Fig. thepsin Kin OCLs gradually increased and reached a peak 4C). at around 6 h after phagocytosis (Figs. 5 and 6A). The high Isolation of LBC from OCLs and Macrophages-After incubation with latex beads for 60 min at 37•Ž, OCLs and macrophages were washed and chased for various periods of time. After lysis by N2 cavitation, the cells were subjected to step-wise sucrose density gradient centrifugation. As shown in Fig. 4D, the fraction obtained from OCLs at the 10/257 sucrose interface consisted of LBC surrounded by continuous and closely apposed membranes. No contamina tion by other organelles was observed in this fraction. The same results were obtained with macrophages (not shown). Kinetic Analysis of the LBC Protein Compositions of OCLs and Macrophages-The protein compositions of LBC isolated from OCLs and macrophages in the course of the endocytic transport pathway were analyzed by immune- blotting with antibodies against various marker proteins followed by densitometric scanning. The LBC level of ca-

Fig. 4. Electron micrographs showing LBC transfer from early to late endosomal elements or phagolysosomes. After in cubation with latex beads at 37•Ž for 1 h, OCLs were washed and chased for 0 (A) or 4 h (B, C). Immediately after phagocytosis, LBC were present at the cell surface of OCLs. At a 4 h chase, LBC were surrounded by electron-dense organelles and localized at or in the vi cinity of the ruffling plasma membranes (B) and in the central area of the cytoplasm of OCLs (C). After lysis of the cells at 4 h after phagocvtosis, the postnuclear supernatant was subjected to stepwise sucrose gradient centrifugation. The LBC fraction isolated at the in terface of the 10-25% sucrose layers was analyzed by electron mi croscopy (D). Arrowheads indicate LBC. Bars indicate 1.0 ƒÊm.

Fig. 3. Distribution and localization of LBC in OCLs. After in cubation with latex beads at 37•Ž for 1 h, OCLs were washed and chased for various time periods. After fixation with 49, paraformal dehyde, the cells were stained for TRAPase and Hoechst33258. At a 0 h chase, latex beads were rapidly phagocytosed and LBC were Fig. 5. Immunoblot analysis of the LBC fraction of OCLs iso preferentially localized on the cell surface of TRAP-positive multinu lated at the indicated times after phagocytosis by antibodies cleated OCLs IA, B). After 4 h of chase, LBC were widely distributed specific for cathepsin K, TRAP, c-Src, and cathepsin D. OCLs throughout the cytoplasm of OCLs (C, D). At 24 h after phagocytosis, were incubated with latex beads for 1 h at 37•Ž and then chased LBC migrated to a more central area of the cytoplasm of OCLs (E, without beads for the indicated times. The LBC fractions isolated by F). Arrows indicate typical LBC localizations. Original magnification sucrose density gradient centrifugation were lysed and subjected to 100. •~ SDS-PAGE and immunoblotting with various antibodies.

J. Biochem. Endocytic Transports in Osteoclasts and Macroph ages 827

level of LBC cathepsin K was maintained throughout the was not detected in the LBC throughout the chase period chase period up to 24 h . However, lysosomal cysteine pro . teinase cathepsin B was barely detectable in osteocla The LBC levels of cathepsin D, a lysosomal aspartic pro sticthro LBCteinase, in both OCLs and macrophages increased in a ughout the chase period (Fig . 6A). In contrast , whiletheLBCtime-dependent manner throughout the chase period cathepsin B level in macrophages increased , but rapidly and then decreased after phagoeytosis the time-course to the peak values differed between the two , cathepsin K cell types. The TRAP level in OCLs increased rapidly in the

Fig. 6. Changes in LBC levels of various marker proteins in times. The LBC fraction isolated lions both cell types were 1, d -~-d OCLs and macrophages during the process of transformation subjected to 01)0 PAGE and immunoblotting with i from early LBC into late endosomal elements or phagolyso Protein levels were quantified by densitometric .scanning, The ,pen somes. Both OCLs and macrophages were incubated with latex and dose eircles indicate the data 16, macrophages and OCLs re beads for 11, at 37•Ž and then chased without . heads for the indicated spectively

Vol. 130. No. 6. 2001 828 E. Sakai et at. early LBC and reached a peak at around 2 h after phagocy were examined. Rab7 was barely detectable in the osteo tosis. This high TRAP level in the LBC was maintained up lastic LBC immediately after phagocytosis, but increasedc to 24 h after phagocytosis. Conversely, TRAP was not de rapidly, reached a peak at around 4 h chase, and then grad tected in the macrophage LBC throughout the chase period ually decreased up to a 24 h chase (Fig. 6B). Conversely, (Fig. 6A). this protein accumulated greatly in the macrophage LBC Since c-Src tyrosine kinases are known to be essential for during the 60-min incubation with latex beads. This accu osteoclastic bone resorption (22-26) and to enhance phago mulated Rab7 in the LBC was decreased exponentially cytosis in macrophages greatly (27), we next examined the during the chase period up to 6 h after phagocytosis. Nei c-Src level in LBC in OCLs and macrophages. The c-Src ther the early endosomal marker protein Rab5 nor the level in the osteoclastic LBC increased rapidly, reached a secretory vesicle marker protein Rab3 was detectable in peak within 2 h after phagocytosis, and then decreased either osteoclastic or macrophage LBC throughout the with time (Fig. 6B). The c-Src protein was no longer detect- chase period (not shown). able in the late LBC after an 18 h chase. Interestingly, c- The lysosomal membrane-associated protein Lamp-1 is Src in the macrophage LBC was not detectable throughout known to be directed to lysosomes after synthesis (39, 40). the chase period (Fig. 6B)- A minor part of Lamp-1 has been shown to be transported Rab GTPases have been shown to be associated with dis to lysosomes via the cell surface as an alternative transport tinct cellular compartments and to function as specific reg pathway (41-43). The Lamp-1 level in the osteoclastic LBC ulators of intracellular transport (for reviews see Ref. 28- increased time-dependently, reaching a plateau value at 31). Therefore, Rab proteins have been used as compart around 6 h after phagocytosis (Fig. 6A). This result is com ment-specific markers as well as tools with which to char patible with that obtained for the macrophage LBC. On the acterize intracellular transport pathways. Among the mem other hand, the Ca2+-dependent phospholipid binding pro bers of this protein family, Rab7 has been shown to localize tein annexin II has been shown to be involved in the regu to late endosomes (32) and to regulate the transformation lation of membrane trafficking events (for reviews see Ref from early endosomes to late endosomes (33. 34) and lyso 44). In addition to serving as a receptor for plasminogen, somes (35). On the other hand, Rab5 localizes to plasma tissue-type plasminogen activator, and plasmin, annexin II membranes and early endosomes (32, 36) and has been is involved in direct vesicle transport to the apical cell sur- shown to control transport from plasma membranes to face of polarized MDCK cells (45). Therefore, we tested sorting endosomes as well as the homotypic fusion of early whether annexin II is involved in the transformation of endosomes (36-38). To gain further insight into the involve early phagosomes to mature phagolysosomes in both cell ment of these small GTPases in the endocytic pathway, the types. Annexin II accumulated at high levels in the early LBC levels of Rab5 and Rab7 in OCLs and macrophages LBC of both OCLs and macrophages during 60-min incuba tion with latex beads. The accumulated annexin II level

Fig. 7. Effects of RANKL on the LBC levels of cathepsin K, Fig. 8. Effects of calcitonin on the LBC levels of cathepsin K, TRAP, and c-Src in OCLs. OCLs were incubated with latex beads TRAP, and Rab7 in OCLs. The cells were incubated with latex for I h at 37•Ž in the presence of 20 ng/ml M-CSF and then chased beads for 1 h at 37•Ž in the presence of 20 hg/ml M-CSF and then without heads for 4 h in the presence or absence of 100 ng/ml chased without beads for 4 h in the presence or absence of 10" M RANKL. An equal number of LBC, as determined by the fluores calcitonin. An equal number of LBC, as determined by the fluores cence intensity at 480 min (excitation at 380 nm), were lysed and cence intensity at 480 non (excitation at 380 non), were lysed and subjected to SDS-PAGE followed by immunoblotting with antibodies subjected to SDS-PAGE followed by immunoblotting with antibodies against each protein (A). The LBC levels of the respective proteins against each protein (A). The LBC levels of the respective proteins were determined using an imaging densitometric analyzer and ex- were determined using an imaging densitometric analyzer and expressed as relative ratios to the non-treated sample (B). pressed as relative ratios to the non-treated sample (B).

J. Biochem. Endocytic Transports in Osteoclasts and Macrophages 829

decreased with time throughout the chase period (Fig . 6B).Eff within a 6 h chase and then gradually decreases. Its disap ects of RANKL and Calcitonin on the Accumulation of pearance may represent the transformation of late endoso Various Molecules in Osteoelastic LBC-The formation and al LBC into phagolysosomes. Similar distribution mprofiles activation of OCLs are now known to be regulated by vari for Rab7 and Lamp-1 strongly suggest that Rab7 plays an ous factors that are systemic, such as Io ,25(OH)2D3 calcito important role in the transformation from early to late nin, and parathyroid hormone , as well as locally produced, endosomal LBC or phagolysosomes. The results are com such as IL-6 and RANKL , by osteoblasts, stroma cells, and patible with previous observations that Rab7 localizes to immune cells (for reviews see Ref 46) . To characterize the late endosomes where it mediates transport from early to osteoclastic LBC further, OCLs were incubated with latex late endosomes (33, 34) or lysosomes (35). In contrast, Rab7 beads for 60 min at 37•Ž in the presence of M-CSF (20 ng/ in the macrophage LBC accumulates maximally within 1 h nil). After phagocytosis, the cells were treated with RANKL of incubation with latex beads at 37•Ž, and rapidly (100 ng/ml) or calcitonin (10-" M) for 4 h at 37•Ž in the decreases during the 6 h chase period, suggesting not only presence of M-CSF (20 ng/ml). The LBC levels of cathepsin its rapid and efficient association with the late endosomal K, TRAP, and c-Src were significantly increased (1 .44 1 LBC during the first 1 h of incubation, but also the rapid 0.09, 1.45 •} 0.08, and 1 .55 0.05-folds, respectively) (Fig . transformation into phagolysosomes. Similarly, changes in 7). The data suggest the stimulation of the transformation the level of cathepsin B in the macrophage LBC shows the of newly formed phagosomes into late endosomes and rapid transformation of early to late endosomal elements or

phagolysosomes. In contrast, the treatment of OCLs with phagolysosomes. Our results suggest that the transforma calcitonin significantly decreased the LBC levels of cathep tion of early to late endosomes or lysosomes in macroph sin K, TRAP, and Rab7 (0.74 •} 0.04, 0.62•} 0 .01, and 0.51 ages is more rapid than in OCLs under the conditions used. 0.08-fold, respectively) (Fig. 8), suggesting the suppres•} On the other hand, Rab5 was not detected in LBC in either sion of the transformation of early endosomes into mature OCLs or macrophages throughout the chase period. How- phagolysosomes. Neither RANKL nor calcitonin caused sig ever, it was found in early LBC formed after incubation nificant changes in the LBC levels of the tested proteins with latex beads for 20 min at 37•Ž (not shown). Since (not shown). Rab5 has been shown to localize to plasma membranes and early endosomes and is thought to control transport from

DISCUSSION plasma membranes to sorting endosomes or the fusion of early endosomes, the isolated LBC, particularly from mac In this paper we describe studies on the endocytic flow of rophages, after 1 h internalization appears to be largely newly formed phagosomes and their fusion with intracellu transformed from early to late endosonral LBC. lar organelles in mouse OCLs and macrophages using theee It has been shown that c-Src plays a critical role in bone latex bead meeeeeeeeeeethod.It is well known that latex bead-loading resorption, and that deletion of the c-Src gene in mice phagosomes serve as useful markers for tracing their endo results in defective osteoclasts and osteopetrosis (25, 26). cytic traffic and characterizing fusion events with intracel However, the signaling pathway regulated by Src in the lular organelles by which LBC acquire specific molecules. endocytic pathways in osteoclasts has not been identified. With this approach, phagosomal subpopulations at various Recent experiments using macrophages also showed that stages of maturation were isolated and functionally charac Src protein kinases greatly enhance phagocytosis but are terized: early endosonral compartments, putative carrier not absolutely essential for the process (27). Our results vesicles, late endosomes, and phagolysosomes. In addition. clearly show the localization of the c-Src protein in osteo as the exocytic vesicle marker Rab3 has not been detected clastic LBC and its association with the transformation of in LBC of either cell type throughout the chase period, the early into late endosomal elements or phagolysosomes. results with this system indicate the characteristics of the However, it is unlikely that c-Src in macrophages is in endocytic transport pathways only volved in this process. After 1 h internalization at 37•Ž, latex beads were In addition to c-Src, the macrophage LBC does not con phagocytosed into the early endosonral elements of OCLs tain cathepsin K or TRAP. Cathepsin K has been shown to found in the cell periphery (Figs. 3 and 4). At 4 h after be confined exclusively to the ruffled border membrane of phagocytosis, the newly formed LBC were found in the osteoclasts and the eroded bone surface, suggesting its role perinuclear region. Considering that the late endosomal in the extracellular degradation of bone matrix proteins (3, and lysosomal marker Lamp-1 and the lysosomal marker 4, 11, 47). The importance of cathepsin K in osteoclastic cathepsin D in the LBC showed maximal concentrations at bone resorption is further supported by observations that 6 and 18 h after phagocytosis, respectively (Fig. 6), the LBC patients with pycnodysostosis have mutations in their fractions between 4 and 6 h mainly consist of late endoso cathepsin K genes (48), and that osteoclasts from cathepsin mal elements, whereas after a 18 h chase, they represent K-deficient mutant mice cannot degrade bone matrix pro phagolysosomal elements. Similar biochemical observations teins (49). Our results show that cathepsin K accumulates were obtained with macrophages. with time in the LBC of OCLs, and that its level is main It is clear from the present study that there are signifi tained after a 24 h chase, indicating that this enzyme is cant differences between the two cell types in the distribu transported to the late endosomal LBC and phagolyso tion and behavior of LBC proteins acquired by fusion with somes where it appears to participate in the intracellular various intracellular organelles during the transformation degradation of the endocytosed matrix proteins. The acqui into mature phagolysosomes. The acquisition pattern of sition profile of TRAP in the LBC is similar to that of Rab7 is strikingly different between OCLs and macro cathepsin K. Since TRAP has been shown to localize to phages. The amount of Rab7 in osteoclastic LBC is very low transcytotic vesicles in osteoclasts and to generate reactive immediately after phagocytosis but increases greatly oxygen species, and thereby, perhaps be able to destroy

Vol. 130 No. 6, 2001 830 E. Sakai et al. endocytosed proteins (50). Together, our present results tivity of human osteoclast cathepsin K. J Biol. Client 271, suggest that. TRAP contributes to the endosonla/lysosomal 12517-12524 degradation of bone matrix proteins. 12. Faust, J., Lacey, D., Hunt, P, Burgess, T.L., Scully, S., Van, G., Furthermore, our results indicate that osteoclastogenesis Eli, A., Qian, Y, and Shalhoub, V. (1999) Osteoclast markers accumulate on cells developing from human peripheral blood regulating factors, such as RANKL and calcitonin, also reg mononuclear precursors. J_ Cell Biochem. 72, 67-80 ulate the association of cathepsin K, TRAP, c-Src, and Rab7 13. Sakai, H., Sofia, T, Kato, Y., and Yamamoto, K (1989) Quanti (Figs, 7 and 8). RANKL has recently been identified as an tation and immunohistochemical localization of cathepsins E osteoclast differentiation factor (51-51), and is known to and D in rat tissues and blood cells. Biochim. Biophys. Acta promote osteoclast bone resorption activity by inducing 991.367-375 actin ring formation (55). Calcitonin is well known to in 14. Wet it, Z. and Color, Z.A. (1972) Plasma membrane synthesis in duce a marked decrease in bone resorption by its receptor the macrophage following phagocytosis of polystyrene latex particles. J. Biol. Chem. 247,2439-2446 mediated signaling pathway (56). The LBC levels of cathep 15. Desjardins, M., Huber, L.A.. Parton, R.G., and Griffiths, G. sin K, TRAP, and c-Src in OCLs 4 h after phagocytosis are (1994) Biogenesis of phagolysosomes proceeds through a se significantly enhanced by treatment with RANK_ In con quential series of interactions with the endocytic apparatus J. trast, calcitonin greatly decreases the association of cathep Col/ Biol. 124, 677--688 sin K. TRAP, and Rab7 with the LBC. It is evident from the 16. Desjardins, M., Celis, J.E., van Meer, G., Dieplinger H., Jahr aus, A., Griffiths, G., and Huber, L.A. 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