Diane M. Biskobing

Pressure regulates osteoclast formation and MCSF expression in marrow culture

One of the forces generated during skeletal loading is hydrostatic pressure. In the work presented here, the ability of increased pressure to influence recruitment of osteoclasts was evaluated. Murine marrow cultures, with pO2 and pCO2 kept constant, were subjected to either control (1.0 atm) or elevated (1.37 or 2.0 atm) hydrostatic pressure. As compared to control, cultures pressurized for 6 days at 1.37 atm formed less osteoclast‐like cells (OCLC) (71 ± 6% of control, P < 0.0001). A similar degree of inhibition occurred in cultures exposed to pressure during days 2–4 only (62 ± 6%), while treatment during days 5–7 failed to inhibit the OCLC number relative to control (99 ± 5%). Delivery of 2.0 atm pressure on days 2–4 generated 52 ± 4% OCLC compared to control. Since macrophage colony stimulating factor (MCSF)‐dependent proliferation of osteoclast precursors occurs during the pressure‐sensitive period, semiquantitative RT‐PCR for MCSF mRNA was performed after 3 days in 1.37 atm (days 2–4). As compared to controls, pressure caused a decrease in mRNA coding for the membrane bound form of MCSF (71.2 ± 4% (n = 25), P ≤ 0.05), while the MCSF RT‐PCR product representing the secreted form showed no consistent change. This lack of response of the soluble MCSF RT‐PCR product was expected, as levels of bioassayable MCSF were not altered by pressure. Extrapolating these data to in vivo conditions suggests that load‐bearing will inhibit the formation of osteoclasts. J Cell Physiol 170:81–87, 1997

Regulation of Murine Osteoblast Macrophage Colony-Stimulating Factor Production by 1,25(OH)2D3

Abstract. Macrophage colony stimulating factor (MCSF) is important for formation of osteoclasts. We investigated the ability of 1,25(OH)2D3 to regulate osteoblast production of MCSF. Mouse calvarial osteoblasts were cultured for 2 days ± 1,25(OH)2D3. Since 1,25(OH)2D3 decreased osteoblast proliferation by 17.6 ± 1% at 10 nM and 11 ± 4% at 1 nM, the effect of growth rate on MCSF secretion was examined. Limiting cell proliferation by serum did not affect MCSF production. 1,25(OH)2D3 (1 nM) increased MCSF production (U/105 cells) maximally by 68 ± 33% (n = 3) with an ED50 for 1,25(OH)2D3 of 5 × 10−11 M. To investigate effects of 1,25(OH)2D3 on MCSF gene regulation, RT-PCR primers were designed to identify the mRNA coding for the membrane-bound isoform of MCSF. Simultaneous RT-PCR of glyceraldehyde-phosphate dehydrogenase (GAP) allowed semiquantitative assessment of MCSF mRNA between treatment groups expressed as the MCSF/GAP RT-PCR product ratio; both MCSF and GAP (+) primers were labeled with 32P-ATP for phosphorimage quantitation. The membrane-bound MCSF/GAP PCR product ratio was not affected by proliferative rate when growth was limited by [serum]. The MCSF/GAP RT-PCR product ratio was dose dependently increased by 1,25(OH)2D3, maximally at 1 nM at 2.2 ± 0.2 = fold (n = 10). 1,25 (OH)2D3 also increased the expression of an RT-PCR MCSF/GAP product ratio which represented the secreted isoform of MCSF. The ability of 1,25(OH)2D3 to pretranslationally regulate expression of membrane-bound osteoblast MCSF may be important in osteoblast:osteoclast interactions.

Macrophage Colony Stimulating Factor Down-Regulates MCSF-Receptor Expression and Entry of Progenitors into the Osteoclast Lineage

Macrophage colony‐stimulating factor (MCSF), although necessary for entry of precursors into the early preosteoclast pathway, inhibits osteoclastogenesis at high doses. To clarify the relationship between MCSF and osteoclast formation, we investigated the effect of exogenous MCSF in murine bone marrow culture. Precursor proliferation and the expression of MCSF‐receptor were examined after 4 days of culture in the presence or absence of accessory stromal cells. In both mixed marrow and destromalized cell cultures, exogenous MCSF dose‐dependently decreased125I‐MCSF binding (by 65 ± 5.0% at 3500 and 87 ± 16.7% at 7000 U/ml, respectively) while enhancing mononuclear cell proliferation after 3 days of exposure (by 2.8‐ and 6.3‐fold, respectively). These effects were maintained 24 h after removal of exogenous MCSF and, as such, likely represented an MCSF‐induced change in MCSF receptor‐bearing cells. Exposure to exogenous MCSF (3500 U/ml) days 2–4 dose‐dependently inhibited tartrate resistant acid phosphatase positive multinuclear cell (TRAP+ MNC) formation counted at the end of day 7, by 64.3 ± 4.1%. This inhibition of TRAP+ MNC formation was preceded by a 92 ± 9% decrease in the expression of carbonic anhydrase II mRNA measurable at 4 days. These results indicate that MCSF promotes proliferation of a population of cells expressing lower cognate receptor sites. Changes in MCSF‐receptor expression appear to modulate the final lineage selection of the pluripotent monoblastic progenitor.

1,25(OH)2D3 and cAMP Synergistically Induce Complement 5a Receptor Messenger RNA

Complement 5a receptor (C5aR) mediates both acute and chronic participation of monocytes in the immune response. In the human U937 monoblast, C5aR is maximally expressed 4 days after treatment with 1,25(OH)2D3 (or cycloheximide) and prostaglandin E2 combined. The authors asked whether these agents altered expression of C5aR messenger RNA (mRNA). Unstimulated U937 cells expressed neither C5aR mRNA nor C5a binding. Complement 5aR mRNA rose 3 hours after prostaglandin E2 application and fell to basal levels by 12 hours. This early rise in C5aR mRNA did not cause an acute rise in C5a binding, which gradually increased between 1 and 4 days. Neither 1,25(OH)2D3 nor cycloheximide induced expression of C5aR mRNA in the absence of prostaglandin E2 but did enhance prostaglandin E2-stimulated C5aR mRNA expression and C5a binding. The authors observed a late increase in C5aR mRNA at day 3 in treated cells. Inhibition of this late rise in mRNA with 5,6-dichlorobenzimidazole riboside attenuated C5a binding by 65%, indicating its importance in the generation of C5a binding sites. The expression of functional C5aR is, therefore, a complex process involving regulation at transcriptional and posttranscriptional levels.