Bone matrix proteins and extracellular acidification: Potential co‐regulators of osteoclast morphology

Abstract

Osteoclasts are signaled by the bone matrix proteins fibronectin (FN), vitronectin (VN), and osteopontin (OPN) via integrins. To perform their resorptive function, osteoclasts cycle between compact (polarized), spread (non-resorbing) and migratory morphologies. Here we investigate the effects of matrix proteins on osteoclast morphology and how those effects are mediated using RAW 264.7 cells differentiated into osteoclasts on FN, VN, and OPN-coated culture dishes. After 96 h, 80% of osteoclasts on FN were compact while 25% and 16% on VN were in compact and migratory states respectively. In contrast, OPN induced osteoclast spreading. Furthermore, osteoclasts formed on VN and FN were two- to fourfold smaller than those formed on OPN in the 21-30 nuclei/osteoclast group. These effects were not due to defects in cytoskeletal reorganization of osteoclasts on VN and FN, demonstrated by the ability of these cells to spread in response to 35 ng/ml macrophage colony stimulating factor (M-CSF). Conversely, osteoclasts on OPN failed to spread when induced by M-CSF. Moreover, the extracellular pH on FN and VN (7.25 and 7.3, respectively) was significantly lower than that on OPN (∼7.4). We further investigated the role of extracellular pH and found that at pH 7.5 the duration of an osteoclast's compact phase was 25.6 min and that of the spread phase was 62.5 min. Reducing the pH to 7.0 increased the frequency of osteoclast cycling by threefold. These results show that matrix proteins play a role in regulating osteoclast morphology, possibly via altering extracellular and intracellular pH.

Fibronectin inhibits osteoclastogenesis while enhancing osteoclast activity via nitric oxide and interleukin‐1β‐mediated signaling pathways

“…VN's effects on osteoclast activity were in line with its inhibitory effects on osteoclast differentiation in that it did not stimulate total resorption compared to FN or OPN and only slightly increased pit formation and resorbed area/osteoclast. The slight increase in pit formation and resorption per osteoclast correlates with our observation that VN increases the ratio of osteoclasts with a migratory morphology [Gramoun et al, 2010].…”

“…Another possible mechanism for increased osteoclast activation on FN involves extracellular pH regulation. Extracellular pH of osteoclasts on FN was indeed lower than cultures on TCP, VN, and OPN [Gramoun et al, 2010]. As low extracellular pH increases osteoclast resorption in vitro, the low pH resulting from FN offers plausible mechanism for the enhancement Fig.…”

Osteopontin Signals through Calcium and Nuclear Factor of Activated T Cells (NFAT) in Osteoclasts

“…Interestingly, these effects were mimicked by RGD-containing peptides and blocked by an antibody directed against the ␣v␤3 integrin. Similarly, it was reported recently that osteoclast-like cells differentiated from murine RAW 264.7 cells on OPN were more spread than cells formed on vitronectin or fibronectin (39). On the other hand, Teti and co-workers (40) reported that authentic rabbit osteoclasts plated on an OPN-coated substratum exhibited less spreading than cells plated on a control surface.…”

Inhibition of osteoclastogenesis by stem cell-derived extracellular matrix through modulation of intracellular reactive oxygen species

“…Even in the presence of RANKL at a high concentration (100 ng/mL), monocytes on ECM still failed to differentiate toward functional osteoclasts, while RANKL at low concentrations, such as 25 ng/mL, was sufficient to induce BMMs to form multinucleated osteoclasts on standard TCPS. In a recent study, it was shown that ECM proteins (fibronectin, vitronectin, and osteopontin) modulated osteoclast morphologies between compact, spread, and migratory shapes, indicating that bone matrix proteins were essential for osteoclast resorption activity [29]. Kajahn et al .…”