Upregulation of Osteopontin by Osteocytes Deprived of Mechanical Loading or Oxygen

Abstract

The pathway(s) by which disuse is transduced into locally mediated osteoclastic resorption remain unknown. We found that both acute disuse (in vivo) and direct hypoxia (in vitro) induced rapid upregulation of OPN expression by osteocytes. Within the context of OPN's role in osteoclast migration and attachment, hypoxia-induced osteocyte OPN expression may serve to mediate disuse-induced bone resorption. Introduction:We have recently reported that disuse induces osteocyte hypoxia. Because hypoxia upregulates osteopontin (OPN) in nonconnective tissue cells, we hypothesized that both disuse and hypoxia would rapidly elevate expression of OPN by osteocytes. Materials and Methods:The response of osteocytes to 24 h of disuse was explored by isolating the left ulna diaphysis of adult male turkeys from loading (n ‫ס‬ 5). Cortical osteocytes staining positive for OPN were determined using immunohistochemistry and confocal microscopy. In vitro experiments were performed to determine if OPN expression was altered in MLO-Y4 osteocytes by direct hypoxia (3, 6, 24, and 48 h) or hypoxia (3 and 24 h) followed by 24 h of reoxygenation. A final in vitro experiment explored the potential of protein kinase C (PKC) to regulate hypoxia-induced osteocyte OPN mRNA alterations. Results: We found that 24 h of disuse significantly elevated osteocyte OPN expression in vivo (145% versus intact bones; p ‫ס‬ 0.02). We confirmed this finding in vitro, by observing rapid and significant upregulation of OPN protein expression after 24 and 48 h of hypoxia. Whereas 24 h of reoxygenation after 3 h of hypoxia restored normal osteocyte OPN expression levels, 24 h of reoxygenation after 24 h of hypoxia did not mitigate elevated osteocyte OPN expression. Finally, preliminary inhibitor studies suggested that PKC serves as a potent upstream regulator of hypoxia-induced osteocyte OPN expression. Conclusions: Given the documented roles of OPN as a mediator of environmental stress (e.g., hypoxia), an osteoclast chemotaxant, and a modulator of osteoclastic attachment to bone, we speculate that hypoxiainduced osteocyte OPN expression may serve to mediate disuse-induced osteoclastic resorption. Furthermore, it seems that a brief window of time exists in which reoxygenation (as might be achieved by reloading bone) can serve to inhibit this pathway.

Sclerostin and the regulation of bone formation: Effects in hip osteoarthritis and femoral neck fracture

“…(40)(41)(42) It also has been established that osteocytes play an important role in directing the tissue renewal process. (43)(44)(45)(46)(47) This study therefore was designed to investigate whether sclerostin (an osteocyte-specific inhibitor of bone formation) is a candidate modulator of the disease effects of osteoporosis and osteoarthritis at the hip. Our results show sclerostin to be a promising explanatory mediator for the decline in osteonal bone formation as osteonal infilling proceeds because the proportion of osteocytes expressing sclerostin increases as the osteon matures.…”

Osteopontin: Role in immune regulation and stress responses

“…In recent years, the role of OPN in other forms of stress such as oxidative and physical stress has been explored as the participation of OPN in various physiological processes expanded from predominantly bone matrix-related functions to a wider area associated with immune regulation, tumorigenesis, autoimmune disease and cell survival (Itoh et al, 2005;Gross et al, 2005;Cao et al, 2007;Wang et al, 2007). That OPN expression is frequently up-regulated in response to various stressors suggests that it mediates the response of the cell or organism to the pertinent stress.…”

“…OPN expression is increased in response to various stressors, including mechanical and oxidative stress (Gross et al, 2005;Maetzler et al, 2007). A connection between stress and immune function has been demonstrated in various experimental paradigms (Nieman and Pedersen, 1999).…”

Molecular pathways mediating mechanical signaling in bone

“…Osteocytes are indeed able to respond to strain in vivo as shown by increased glucose-6 phosphate dehydrogenase activity (Skerry et al, 1989), or earlier response of c-fos mRNA (Inaoka et al, 1995) after loading. As well, unloading causes osteopontin expression in osteocytes (Gross et al, 2005). Dentin matrix protein (DMP1), which is a secreted matrix protein expressed in late osteoblasts and osteocytes, has been shown to increase in osteocytes after tooth movement in the jaw (Gluhak-Heinrich et al, 2003).…”