Trafficking of Matrix Collagens Through Bone-Resorbing Osteoclasts

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Science  11 Apr 1997:
Vol. 276, Issue 5310, pp. 266-269
DOI: 10.1126/science.276.5310.266


An intracellular pathway for proteins liberated from mineralized matrix during resorption was identified in osteoclasts. Analysis by confocal microscopy of sites of active bone resorption showed that released matrix proteins, including degraded type I collagen, were endocytosed along the ruffled border within the resorption compartment and transcytosed through the osteoclast to the basolateral membrane. Intracellular trafficking of degraded collagen, as typified by the resorbing osteoclast, may provide the cell with a regulatory mechanism for the control of tissue degradation.

Osteoclasts are multinucleate bone cells with the capacity to degrade the extracellular matrix of the skeleton by the process of bone resorption, thus participating in the homeostasis of bone and calcium (1). Osteoclasts resorb mineralized tissues after a series of cellular polarization events (2). Cytoskeletal rearrangement creates an F- actin–rich structure, the tight seal, that encloses a specialized secretory membrane, the ruffled border. Protons and proteases cross the ruffled border and degrade bone matrix through demineralization and proteolytic activity, with calcium and type I collagen fragments being liberated into a resorption compartment beneath the cell. These products reach the extracellular space, where their levels correlate with bone resorption activity (3).

It has been assumed that degraded bone matrix leaves the resorption site by leakage from under the osteoclast during cell migration or is released en masse at the termination of the resorption cycle when the resorption compartment is disassembled. Alternatively, an enclosed resorption site could be maintained if there were intracellular pathways to transport degraded bone through the osteoclast to the extracellular space, a process for which we provide three lines of evidence. Intracellular trafficking of mineralized matrix components was assessed in human osteoclasts [isolated from giant cell tumors of bone (4)] in a culture model of bone resorption that uses dentine substrate (5), and in osteoclasts resident in bone. Matrix proteins were localized at sites of resorption (6) by immunostaining and confocal microscopy (7, 8).

Trafficking of matrix proteins through resorbing osteoclasts was identified with the use of a labeled dentine substrate (Fig.1). Surface proteins in the dentine slices were biotinylated before osteoclast attachment and resorption and then detected with streptavidin-fluorescein (9). Resorption of matrix was seen as a loss of the surface biotin label and hence as an absence of specific staining (Fig. 1E). Many of the osteoclasts formed resorption tracks as they tunneled through the dentine and were identified at the end of the tracks by their characteristic F-actin staining pattern (Fig. 1, D and F). These ring- or arc-shaped structures are associated with the resorption compartment (2), and in tunneling osteoclasts, they indicate the position of the tight seal and ruffled border, which appose the surface undergoing resorption (Fig. 1F). Matrix components liberated into the resorption compartment (Fig. 1E) were detected within the ruffled border of the osteoclast (Fig.1F). Many (92%; n = 51) of these osteoclasts had a distinct pattern of intracellular matrix proteins in which the proteins were localized within a cytoplasmic pool in the body (Fig. 1B) and toward the top of the osteoclast (Fig.1A). These various staining patterns indicate that released matrix proteins were endocytosed along the ruffled border within the resorption pit and transcytosed through the osteoclast to the basolateral membrane facing the extracellular space, away from the resorbing surface.

Figure 1

(A to F) Trafficking of biotinylated dentine matrix proteins through resorbing osteoclasts. Human osteoclasts were cultured on biotinylated dentine slices for 24 hours (6, 7). The cells were fixed and permeablized before fluorescent immunostaining and confocal microscopy (8). Sectional images (1-μm sections) from the x-yplane (above view) are shown for a typical resorbing osteoclast containing dentine matrix proteins (n = 51). The false colors of fluorescence represented are green (bone matrix proteins) and red (F-actin). Images are of sections taken at the following depths from the top of the osteoclast: (A) at 4 μm (shows the top of cell); (B) and (C) at 10 μm (showing the basolateral body of the cell); and (D), (E), and (F) at 20 μm (showing the cell at the dentine surface). The single fluorescent images show in (C), the spread cell periphery above the dentine surface; in (D), the ruffled border and location of the active resorption site that extended beneath the cell into the resorption pit (arrow, dense red F-actin arc structure); and in (E), a zone of liberated bone matrix proteins (small arrowheads) and loss of bone matrix to form the resorption pit (black). Double fluorescent images in (A) and (B) show the basolateral distributions of bone matrix proteins in the osteoclast (small arrowheads). Image (F) represents the images of (D) and (E) superimposed and reveals that the liberated matrix proteins are present within the ruffled border. The direction of osteoclastic resorption [broad arrowhead in images (B) and (F)] is indicated by the track of resorption left behind the osteoclast (pit edge shown by small arrows). The resorption pit extended 20 μm below the dentine surface through which the osteoclast maintained close contact with the resorbed surface of dentine [image (D), fine F-actin ring indicating the osteoclast periphery in the resorption pit]. Original magnification ×600.

Previous studies have suggested that osteoclasts can endocytose protein (10, 11). To investigate osteoclast endocytosis of matrix collagen, we probed the resorption cultures with an antibody to native type I collagen (Fig. 2, A to D). Intracellular localization of collagen in osteoclasts indicated that endocytosis of collagen from the extracellular dentine matrix was occurring because osteoclasts do not synthesize type I collagen (12). Confocal microscopy was used to generate images of 1-μm sections through the resorption cultures in both horizontal (x-y plane, above view in Fig. 2, A and B) and vertical planes (z-x plane, lateral view in Fig.2, C and D). Type I collagen was located along the unresorbed bone surface and in the resorption pit. Collagen was seen only in the exposed regions of the pit where it had proceeded in a lateral direction and thus left a trail of resorption behind the osteoclast (Fig. 2D). Collagen was not detected throughout the dentine slice because it is impervious to antibody below a depth of 5 μm, a depth comparable with that achieved by biotinylation. Endocytosis of bone matrix collagens was identified in 97% of resorbing osteoclasts (n = 116). Generally, endocytosed collagen was localized throughout the cytoplasm of the osteoclast, with an additional focus toward the basolateral membrane, which faced the extracellular space (Fig. 2D). The pattern of intracellular collagen was comparable with that obtained for the biotinylated dentine matrix proteins (Fig. 1) and indicated that endocytosis and transcytosis of dentine matrix collagen was occurring through the osteoclast during resorption.

Figure 2

(A to D) Trafficking of collagen in resorbing osteoclasts. Osteoclasts were cultured on dentine for 24 hours (6, 7) and processed for immunostaining of collagen and confocal microscopy analysis (8). Sections (1 μm thick) of two of 116 osteoclasts assessed for type I collagen localization are shown. (A) shows anx-y section (above view) taken at the bone surface, (B) shows an x-y section taken 4 μm above the bone surface through the top of the osteoclast, and (C) and (D) showz-x sections (lateral views) for the cells in (A) and (B), respectively, taken through the center (broken lines denote the bone surface). The edge of the resorption pit is indicated by arrows; the tops of the resorbing osteoclasts are starred. Osteoclasts maintained close contact with the resorption face [arrows in (C) and (D)]. The direction of resorption (broad arrow) was indicated by the track left behind the osteoclast [pit edge is indicated by arrows in (A) to (D)]. Original magnification ×600. (E) Localization of collagen in osteoclasts within osteoclastoma tissue. Sections (10 μm thick) of osteoclastoma were immunostained and analyzed by confocal microscopy (8). Osteoclasts were identified by an antibody to the integrin αvβ3 which is highly expressed in osteoclasts (22). A 1-μm x-y section at 5-μm tissue depth is shown. False colors of fluorescence are green for αvβ3 on osteoclasts and red for type I collagen in the surrounding cells, and yellow reveals colocalization of collagen and αvβ3 at the osteoclast surface. Most osteoclasts are not in contact with bone and represent cells in a nonresorbing state. These had not endocytosed collagen, even though large amounts of collagen were visible in the surrounding tissue. Original magnification ×400. (F to H) Localization of collagen in osteoclasts in fetal bone. Collagen was identified in resorbing osteoclasts in frozen sections of human fetal bone. (F) is a nonconfocal phase image (osteoclast indicated by an arrow on bone trabeculum, open star). (G) and (H) are 1-μmx-y sections at 5-μm tissue depth for αvβ3 (red) and type I collagen fragments (green). Original magnification ×600.

The trafficking of bone matrix proteins and collagens appeared to be initiated during the bone resorption process. These proteins were not detected in nonresorbing osteoclasts when examined in short-term cultures (13), nor in cultures where bone resorption was inhibited (14); nor were they detected in vivo in osteoclastoma tissue that had been separated from bone matrix and had no endogenous resorptive activity (Fig. 2E). However, intracellular collagen was detected in osteoclasts in contact with bone surfaces from human fetal tissue, which were exhibiting active trabecular resorption (Fig. 2, F to H) (15).

Bone collagens are extensively degraded by the action of collagenolytic enzymes during resorption (16). The resultant release of type I collagen fragments into the extracellular space and subsequent detection in plasma and urine provide a clinical measurement of bone resorption (17). The localization of collagen was assessed further in the human resorption cultures with antibodies that specifically recognize degraded collagen (18), and in resorbing rabbit osteoclasts (13). Degraded collagen was specific for the resorption site (Figs. 3 and4). Osteoclastic resorption was assessed on biotinylated dentine substrates, which aided the identification of the resorption sites through surface loss of the biotin label (Fig.3A). An estimated 90% of type I collagen fragment immunostaining was within the resorption sites (Fig. 3, B and C,x-y tilted above view images, and Fig. 4A,z-x lateral view image). Intracellular localization in the osteoclast was comparable with that seen for type I collagen (Fig. 2). Degraded collagen lined the resorption pit. Additionally, collagen degradation was detected at a low level on the dentine surface surrounding the periphery of resorption pits, under the cytoplasmic skirt of the osteoclast (Fig.3B). Thus, collagen degradation appears to be contained within the site of osteoclastic bone resorption and so provides evidence that the resorption compartment functions as an enclosed structure.

Figure 3

(A to C) Trafficking of type I collagen fragments from dentine matrix through resorbing osteoclasts. Human osteoclasts were cultured on biotinylated dentine and processed as in Fig. 1 (6-8). Computer-generated compression images are shown for a typical resorbing osteoclast containing type I collagen fragments endocytosed from the dentine matrix (n = 105). Forty 1-μm x-ysections were taken through the resorption site and compressed into a single image and displayed on a 30% incline. False colors of fluorescence represented are green (biotinylated matrix proteins) and red (type I collagen fragments). The single fluorescent image in (A) shows the dentine matrix and resorption site (black), and the image in (B) indicates the localization of collagen fragments at the resorption site within two interconnected pits (the one on the right is indicated by an arrow). An osteoclast is located in the pit on the left (its spread cell periphery above the dentine surface is denoted by open arrows). Collagen fragments were observed in the pits and within the resorbing osteoclast. (C) is a superimposed image of (A) and (B) and shows that the type I collagen fragments are concentrated within the site of bone resorption. Original magnification ×600.

Figure 4

(A to C) Cell surface localization of type I collagen fragments and biotinylated dentine matrix proteins in resorbing osteoclasts. Osteoclasts were cultured on unlabeled and biotinylated dentine for 24 hours and processed as in Fig. 1 (6-8). Lateral views are shown for 1-μmz-x sections taken through sites of resorption (n = 105). (A) and (B) show the localization of type I collagen fragments, (C) shows the localization of biotinylated matrix proteins, and (B) and (C) are nonpermeablized and show sites on the external surface of the osteoclast. Pits [arrows in (A) to (C)] extended below the dentine surface [indicated in (A) and (B) by horizontal broken lines; open arrows in (C) show the surface biotin label]. The tops of resorbing osteoclasts are starred. The cell membrane of osteoclasts are outlined in (B) and (C) by curved broken lines. Below the dentine surface osteoclasts are in close contact with the resorption site (curved broken line with arrows). Above the dentine surface, type I collagen fragments formed a basolateral “cap” at the cell surface in 80% of actively resorbing osteoclasts [image (B)]. Similar structures were seen with biotinylated bone matrix proteins in 15% of resorbing osteoclasts [image (C)]. Collagen fragments were absent from the unresorbed surfaces [horizontal broken lines in (A) and (B)]. Collagen fragments were also seen in resorbing osteoclasts with a focus in the basolateral aspect [image (A)]. The direction of osteoclastic resorption [broad arrow in (A)] was indicated by the track of resorption left behind the osteoclast. Original magnification ×600.

During bone resorption, the osteoclast maintains its ruffled border in close contact with the bone resorption surface (19), which suggests that, as the degraded bone matrix is liberated, it is continually cleared from these enclosed resorption sites. Thus, continual trafficking of the excavated bone matrix through the resorbing osteoclast to the extracellular space would seem essential if the cell is to maintain and contain bone resorption while resorbing further into the bone matrix. In support of this continual clearance of degraded matrix, biotin-labeled matrix proteins were not found in 81% of osteoclasts (n = 105) that were trafficking degraded collagens (Fig. 3C). These osteoclasts had resorbed through the 5-μm depth of the surface biotin label, thus suggesting that the initial endocytosed bone matrix proteins had been rapidly cleared through the osteoclast as resorption proceeded deeper into the dentine slice, typically 20 to 30 μm.

The intracellular localization of matrix proteins, type I collagen, and collagen fragments showed similar distribution patterns in resorbing osteoclasts, with a particular focus toward the basolateral region of the cell (Figs. 1, B and F, 2D, and 4A). Together, these results demonstrate that a general transcytotic pathway exists for degraded matrix proteins in resorbing osteoclasts, which appears to be analogous to the transcytosis of proteins across epithelial and endothelial cellular barriers (20). Transcytotic routes to the basolateral membrane have been implicated in bone-resorbing osteoclasts in studies with viral antigenic markers (11). These studies identified a central zone at the top of the basolateral membrane of the osteoclast that appeared to be a specialized site of exocytosis for resorbing osteoclasts, though this was not formally proven. Akin to this exocytotic site, type I collagen fragments and biotinylated matrix proteins were at the basolateral cell surface, forming a “cap” on a majority of resorbing osteoclasts (Fig. 4, B and C). The basolateral “cap” was absent from 15 to 20% of resorbing osteoclasts that had intracellular trafficking of degraded bone matrix proteins and collagens. This suggests that endocytosed and transcytosed degraded bone matrix collagens are released into the adjacent extracellular space from this region of resorbing osteoclasts.

The ruffled border in a resorbing osteoclast has been described as functionally equivalent to that of a secondary lysosome, being responsible for degradation of bone matrix within the confines of the resorption compartment (21). Intracellular trafficking of products resulting from tissue degradation may provide cells with a mechanism that enables them to monitor and hence control proteolytic activity; for the osteoclast this in turn would regulate the bone resorption process itself. Pharmacological intervention into this intracellular trafficking pathway for degraded extracellular matrix proteins may provide an approach to regulate tissue breakdown in disease.


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