Upregulation of osteopontin by osteocytes deprived of mechanical loading or oxygen.
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.


BACKGROUND
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.


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 = 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 = 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 hypoxia-induced 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.
Gross
Ted S
TS
Orthopaedic Science Laboratories, Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, WA 98104-2499, USA. tgross@u.washington.edu
King
Katy A
KA
Rabaia
Natalia A
NA
Pathare
Pranali
P
Srinivasan
Sundar
S
D000818:Animals; D015153:Blotting, Western; D001862:Bone Resorption; D002465:Cell Movement; D002478:Cells, Cultured; D002633:Chemotaxis; D000860:Hypoxia; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D010010:Osteoclasts / Q000378:metabolism; D010011:Osteocytes / Q000378:metabolism*; D053495:Osteopontin; D010100:Oxygen / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000096:biosynthesis*; D013997:Time Factors; D014422:Turkeys; D015854:Up-Regulation
D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D053495:Osteopontin; D011493:Protein Kinase C; D010100:Oxygen