In this report we investigated the role of the newly described TNF-related protein, OPGL, in OC activation. Previously, Lacey et al. (1998)
and Yasuda et al. (1998b)
identified OPGL/ODF as the long sought OC differentiation factor. Direct expression cloning was used independently by the two groups to identify OPGL/ODF as the ligand for OPG/OCIF. OPG is expressed only as a soluble form and is now believed to act as a soluble decoy receptor to regulate the action of OPGL on differentiation of OCs. The data presented in the two reports provide strong evidence that OPGL acts directly on a population of OC progenitors, and together with CSF-1 induces terminal differentiation into mature, active OCs. Our data also showed that OPGL activated mature OCs to resorb bone in vitro (Lacey et al., 1998
), and recent work supports our previous results (Fuller et al., 1998
In this report we have shown that OPGL or agonist antibodies to its receptor, RANK, act directly on fully differentiated, mature OCs inducing individual OCs to undergo a rapid rearrangement of their actin cytoskeleton into actin rings and to perform multiple cycles of bone resorption as seen by scanning EM. The data demonstrate that many individual OCs are induced to perform multiple cycles of resorption during the assay period, but we also find that more of the OCs in the cultures appear to be activated as suggested by an increase in the number and density of single, isolated resorption events generated by OPGL treated OCs.
Because PTH has been shown to induce OPGL expression in primary osteoblasts (Yasuda et al., 1998b
) and osteoblastic cell lines, (Fuller et al., 1998
), it would be interesting to more carefully quantify the relationship between the size and spatial distribution of the resorption events as reported by Murrills et al. (1990)
. They showed that PTH treatment of rat OC cultures primarily increased the number of resorption foci (defined as resorption lacunae lying within an area of bone covering 1/116th of a bone slice), suggesting that more of the OCs were activated in PTH treated cultures (presumably due to stimulation of OPGL expression by osteoblastic cells present in the cultures). Some of the multiple excavations we observe, especially groups of smaller resorbed areas, may be generated by single large OCs containing multiple actin rings (e.g., see Fig. A, bottom right), or by small OCs performing multiple resorption cycles in a focal region of the bone slice as the result of OPGL activation.
In addition to the quantitative effects of OPGL on OCs, we also found that the resorbed bone surfaces were quite different: while resorbed areas generated by untreated OCs are fairly smooth and usually single, areas resorbed by OPGL activated OCs are frequently multiple connected excavations, which expose numerous collagen fibrils. These continuous excavations appear to be very similar to those described by Chambers et al. (1984)
resulting from OC resorption on anorganic bone (hydrazine treated) compared with the intermittent resorption that occurred on whole bone. How these two observations might be related is unknown, but may reflect different OC residence times at the resorption site and/or the probability that the OC will migrate to a new location under the different conditions. It is possible that OC movement and cycle reactivation is mediated by calcium released during each resorption cycle (Malgaroli et al., 1989
; Zaidi et al., 1989
; for review see Hall and Chambers, 1996
We found that OPG inhibited the activation of isolated OCs by OPGL in vitro, however basal OC activity was not significantly decreased by OPG alone. However, OPG caused an inhibitory trend in several independent experiments. This might be due to the presence of endogenous OPGL in the system, (either from the cell preparation, or the serum) that excess OPG would inhibit. Dempster reported similar observations at a recent meeting (Dempster et al., 1998, The American Society for Bone and Mineral Research. Abstract F087. Bone.
23:S432). Nonetheless, even in the presence of excess OPG, OCs retain a low level of basal resorbing activity suggesting that something other than OPGL is responsible for regulating the basal level of OC resorption. We cannot, however, rule out the possibility that some residual ex vivo OC activity is due to prior OPGL exposure of the OCs in vivo. The marked stimulation of bone resorption in these cultures by OPGL does not appear to be mediated by increases in the number of multinucleate OCs as their numbers did not significantly change after the various treatments. In contrast, Fuller et al. (1998)
have recently reported that OPGL (TRANCE) acts as an OC survival factor in vitro, as has been well documented for CSF-1 (Fuller et al., 1993
). This apparent discrepancy may be explained by the fact that our OC survival measurements were performed on OCs cultured on bone slices, while those of Fuller et al., were on OCs cultured on untreated glass coverslips (even in their hands, OCs plated on bone do not require OPGL for survival, see Fig. , Fuller et al., 1998
). It seems likely that under certain conditions, OPGL can act as a survival factor. Finally, in contrast to the action of OPGL on OC differentiation, activation of mature OCs on bone, or stimulation of actin ring formation on glass by OPGL occurs in the absence of added CSF-1.
Initial observations that OPG treatment of growing mice induced a very rapid (3 d) increase in bone density, led us to consider that OPG might act to inhibit OC activity in addition to being an antagonist of OC differentiation (Simonet et al., 1997
). Furthermore, OPGL caused hypercalcemia within 2 d in vivo, possibly due to the activation of preformed OCs (Lacey et al., 1998
). By investigating mature OCs in culture and performing very short-term in vivo experiments, we have tried to distinguish between the role of OPGL on OC differentiation and the action of OPGL on stimulating mature OCs in culture, and preexisting OCs in vivo to resorb bone: OPGL clearly plays a role in both OC differentiation (Lacey et al., 1998
; Yasuda et al., 1998b
) and OC activation in vitro (Lacey et al., 1998
, Fuller et al., 1998
and present data). The interpretation of the 1 h in vivo treatment of mice with OPGL is complex, as we cannot rule out the kidney as the source of the increase in blood ionized calcium. However, we found that mice maintained on a low calcium diet for 48 h still show a significant and dose-dependent elevation in blood ionized calcium in response to OPGL (see Materials and Methods; data not shown), thus ruling out the gut absorption as the source of calcium. Given the robust and rapid activation of OCs in vitro by OPGL as evinced by both bone resorption and actin ring formation shown here, it seems most likely that OC activation is involved in vivo as well.
OPGL is identical to RANKL/TRANCE, (Wong et al., 1997
), and it has been previously suggested that RANK is its receptor on OC progenitors (Lacey et al., 1998
; Yasuda et al., 1998b
). Recently Hsu et al. (1999)
and Nakagawa et al. (1998)
provided direct evidence that OPGL exerts its activity on OC progenitors via its receptor RANK. We show here that a monospecific antibody to RANK bound to isolated multinucleate OCs demonstrating that RANK is expressed at the surface by mature OCs. In support of our result, Hsu et al. (1999)
recently demonstrated that RANK mRNA is expressed by mature OCs in situ. The anti-RANK polyclonal antibody was found to activate OCs as evinced by OC polarization and formation of actin rings, in an apparently not independent manner to OPGL. The most likely explanation is that the anti-RANK antibody was acting as an agonist by binding RANK, causing receptor aggregation and signal transduction (see Nakagawa et al., 1998
). Together these pieces of evidence implicate RANK as the relevant receptor for OPGL mediated cytoskeletal rearrangements and osteoclast activation.
At this time, it is unknown how liganding RANK leads to cytoskeletal rearrangement and ultimately to activation of bone resorption in the OC, however, several signaling molecules have been specifically implicated in the cytoskeletal rearrangements associated with OC activation that may also play a role in OPGL activation of OCs. pp60c-src
is clearly a central and key component involved in activation of mature OCs. pp60c-src
is highly expressed in OCs (Horne et al.; Tanaka et al., 1992
) and c-src−/− knockout mice exhibit profound osteopetrosis (Soriano et al., 1991
) due to an inability of c-src−/− OCs to become polarized, form actin rings or ruffled borders; all of which are necessary for bone resorption (Soriano et al., 1991
; Boyce et al., 1992
; Lowe et al., 1993
). More recent evidence links the engagement of αv
integrin via pp60c-src
(translocation and activation) to PI3 kinase activation (Hall et al., 1995
; Hruska et al., 1995
; Nakamura et al., 1995
; Chellaiah and Hruska, 1996
; Hruska et al., 1996; Lakkakorpi et al., 1997
; and Nakamura et al., 1997
), and association with the F-actin capping/severing protein, gelsolin (Chellaiah et al., 1998
). Thus for the first time, a specific cytoskeletal protein (gelsolin) and mechanism (reversal of actin capping to support further F-actin polymerization) have been implicated in OC activation by receptor engagement and cell attachment. The stimulation of RANK by OPGL appears to enhance cytoskeletal rearrangements beyond those induced by OC attachment and integrin engagement, and leads to marked stimulation of bone resorption. It will be very interesting to determine if further enhancement of this signaling pathway involving pp60c-src
and PI3 kinase or a completely separate path is responsible for OPGL-RANK–induced actin ring formation and OC activation. Recent data from several groups, (Darnay et al., 1998
; Wong et al., 1998
; Hsu et al., 1999
) suggest that signaling through RANK is mediated by binding to TRAF (TNFR-associated factor) family members. Data from Hsu et al. (1999)
further suggests that JNK activation downstream of RANK/TRAF interactions may be important for OC-like cell differentiation. Events downstream of OPGL-RANK–mediated OC cytoskeletal changes remain to be investigated.
In summary, OPGL binds to individual mature OC- inducing cytoskeletal changes indicative of OC activation and stimulates multiple spatially associated cycles of robust bone resorption in vitro. These effects of OPGL are very selective as they can be inhibited by the natural soluble decoy receptor, OPG, or mimicked by agonistic antibodies to the OPGL receptor, RANK. In addition, OPGL given intravenously induces a rapid increase in blood ionized calcium in mice suggesting that preexisting OCs are activated by OPGL in vivo. Based on these many pieces of evidence, we conclude that in addition to its role in OC differentiation, OPGL is a potent and direct regulator of OC activity in vitro and in vivo.