In 1990, Udagawa et al. first generated bone fide osteoclasts in culture by coculturing marrow macrophages and osteoblast precursors (24
). The discovery that RANKL, produced by osteoblasts and their progenitors, is the key osteoclastogenic cytokine established that marrow stromal cells are essential for physiological osteoclastogenesis (25
The alternative concept that osteoblast recruitment to sites of bone remodeling is mediated by the osteoclast is longer-standing yet suffers from a paucity of mechanistic insights (26
). Two general theories of osteoclast regulation of the osteoblast present themselves. Specifically, the osteoclast itself may be the source of unidentified, osteoblast-trophic molecules. Buttressing this argument is the fact that certain forms of osteopetrosis, such as c-src deficiency, are characterized by abundant, albeit dysfunctional, osteoclasts, yet bone formation is accelerated (27
). In other iterations of the disease, such as that due to lack of c-Fos, osteoclastogenesis is arrested and bone formation dampened (28
). An equally compelling argument holds that the osteoclast mobilizes matrix-residing growth factors, such as TGF-β and IGFs, which, in turn, target and activate the osteoblast (30
). Regardless of mechanism, the sequential tethering of osteoclastic and osteoblastic activity has important clinical implications. For example, antiresorptive agents, such as bisphosphonates, are attended by suppressed bone formation, a phenomenon that may eventuate in adynamic bone and its structural consequences (31
). Similarly, the bone anabolic properties of PTH are diminished when it is administered with an antiresorptive drug (7
Prolonged GC therapy leads to yet another example of structurally inferior, adynamic bone, indicating that repressed osteoclast function may contribute to diminished formation. These observations are in keeping with the suggestion of Weinstein that steroid-induced osteoporosis reflects combined inhibition of both the osteoblast and osteoclast (1
) and indicate that, as in other states of adynamic bone, GC-mediated inhibition of osteoclast function retards remodeling and, hence, dampens osteogenesis.
The issue as to whether GCs impact osteoclastic bone resorption has been controversial. The capacity of these steroids to suppress intestinal absorption and renal tubular reabsorption of calcium is consistent with a scenario of hormonally stimulated bone degradation (32
). On the other hand, PTH levels are probably not increased in most GC-treated patients, and their skeletal response to PTH-suppressing agents, such as vitamin D, is modest (32
). Similarly, GCs attenuate production of sex steroids, low levels of which, in other circumstances, promote osteoclastic bone resorption (33
). Hormone replacement therapy, however, does not retard GC-induced bone loss (34
). Therefore, if GCs impact the osteoclast, they are likely to do so directly or within the context of locally produced osteoclast-regulating factors such as RANKL or M-CSF.
Our studies of the effects of GCs on various phases of osteoclast differentiation and function benefited from the availability of mice conditionally disrupting the GR in the entire BMM/osteoclast lineage, which permitted us to determine whether individual biological events are specific and GR mediated. These mutant mice appear indistinguishable from WT for at least 2 months, indicating that GC-responsive osteoclasts are not required for normal skeletal development. Probably reflecting the short duration of treatment, histomorphometrically measured bone mass of WT and GRoc–/– mice, following 14 days of steroid exposure, was similar (data not shown).
We found that DEX regulates osteoclast precursor proliferation but does so in a differentiation-dependent manner. Thus, uncommitted BMMs or those stimulated only by M-CSF to develop into mature non-osteoclastogenic macrophages, underwent divisional arrest in the presence of DEX. IL-1α, which alone is not osteoclastogenic (35
), also failed to prevent DEX-suppressed BMM proliferation. In contrast, when the cells were maintained in osteoclastogenic medium containing M-CSF plus RANKL or TNF-α, the steroid no longer dampened proliferation. Thus, commitment to the bone-resorptive phenotype protects macrophages from the antiproliferative effects of GCs. Failure of DEX to suppress RANKL-treated osteoclast precursors was mirrored by unaltered activation of ERKs, which mediates replication of the cell.
Weinstein et al. report that GCs prevent apoptosis of the mature osteoclast (14
), which we confirm. The steroid’s antiapoptotic properties are not attended by Akt activation, indicating that an alternative survival pathway is extant. We also note that the antiapoptotic effect requires only short exposure to the steroid in the latter phase of osteoclast differentiation. Despite their prolonged longevity, however, GC-treated osteoclasts have markedly suppressed bone-resorptive activity in vitro and in vivo. GCs induce RANKL and M-CSF expression and blunt synthesis of the osteoclast-inhibitory molecule osteoprotegerin (12
). These observations, taken with the normal proliferation of DEX-treated BMMs committed to the osteoclast phenotype, and the drug’s antiapoptotic effect on the fully differentiated polykaryon, suggest the steroid would accelerate bone resorption in vivo. We found, however, that while the number of osteoclasts in PTH-stimulated WT mice treated with DEX mirrors the number in those that have not received the steroid, their global bone-resorptive activity is suppressed in a GR-mediated fashion. This paradox of suppressed bone resorption in the face of unaltered osteoclast number indicates that the stimulatory effects of GCs on osteoclast survival are obviated by direct inhibition of the differentiated cell’s capacity to degrade bone.
The osteoclast is characterized by a unique cytoskeleton, which undergoes continuous reorganization with different phases of the resorptive cycle (11
). We find that DEX, in low nanomolar concentrations, disarranges the osteoclast cytoskeleton, yielding cells that fail to spread and ineffectively resorb mineralized matrix. Such features are characteristic of a variety of resorptive disorders, such as absence of the cytoskeleton-regulating proteins, c-src (13
), or the αvβ3 integrin (36
). These in vitro GC-induced events are reflected in vivo by small, irregular osteoclasts that fail to attach to or normally degrade bone and lack the cell’s key resorptive organelle, its ruffled membrane.
Skeletal degradation occurs in an acidified microenvironment between the osteoclast and the bone surface that is isolated from the general extracellular space by an actin ring (11
). Insufficient actin ring generation, in a number of circumstances, is accompanied by failure of the osteoclast to spread or adequately resorb bone. We describe what we believe to be a novel and rapid assay for actin ring formation in osteoclasts under the influence of individual cytokines. Interestingly, DEX did not alter the capacity of RANKL, TNF-α, or IL-1α to establish actin rings in committed osteoclast precursors but substantially suppressed that stimulated by M-CSF. While M-CSF is classically viewed as a survival and proliferative cytokine for osteoclast precursors, it also exerts cytoskeletal effects via a signaling pathway shared by the αvβ3 integrin (37
). Thus, the spreading defect of αvβ3 integrin–deficient osteoclasts is rescued by high-dose M-CSF.
The osteoclast cytoskeleton is modulated by a series of small GTPases including RhoA (38
) and Rac (23
) that transit to their GTP bond state under the influence of guanine nucleotide exchange factors (GEFs). RhoA and Rac are activated in the osteoclast by αvβ3 integrin occupancy or M-CSF, both of which induce the cell-specific GEF isoform Vav3 (23
). Inhibition of RhoA activity or genetic deletion of Rac (22
) or Vav3 (23
) arrests osteoclastic bone resorption, and we found that DEX prevents M-CSF–mediated induction of the 3 entities. Hence, GCs suppressed bone resorption by disrupting the cytoskeleton of the mature resorptive cell in an M-CSF/Vav3/RhoA/Rac-dependent manner.
GCs impact cells by genomic and nongenomic mechanisms. The steroid’s nongenomic effects occur within seconds to minutes and are mediated by the GR or by other means such as G protein–coupled receptors (39
). Because DEX-mediated suppression of Vav3 activation requires 16 hours, a genomic locus of action is likely. Whether this genomic effect involves direct DNA binding of the GR or represents GR associating with an intermediary DNA-interacting protein remains to be determined. In any event, our findings provide what we believe to be a novel paradigm for the pathogenesis of a common and often devastating form of osteoporosis that to date has remained largely refractory to therapeutic intervention.