Reduced expression of platelet endothelial cell adhesion molecule-1 in bone marrow cells in mice after skeletal unloading.

UNLABELLED: One week of tail suspension significantly decreased the expression of PECAM-1 in mouse tibial bone marrow cells but not those of a number of other vascular factors. Anti-PECAM-1 antibody suppressed both ALP+ CFU-f formation and ALP production under co-culture of the osteoblastic cell line and the PECAM-1+ endothelial cell line. This study suggests that the reduced ALP activity after skeletal unloading is related to downregulation of PECAM-1 expression in bone marrow cells in mice. INTRODUCTION: Vascular factors play a role in bone development and regeneration. We tested the hypothesis that skeletal unloading reduces osteogenic potential by inhibiting the molecules related to angiogenesis and/or vasculogenesis in bone marrow cells. MATERIALS AND METHODS: Eight-week-old male mice were assigned to three groups after acclimatization for 1 week: ground control (GC), tail suspension (TS), and reloading after 7-day TS (RL). Bilateral tibial and humeral samples were used for analyses. MC3T3-E1, a mouse osteoblastic cell line, and EOMA and ISOS-1, mouse endothelial cell lines, were also used. RESULTS: Flow cytometric analysis revealed that 7-day TS significantly decreased the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) in tibial bone marrow cells, but not those of angiopoietin-1, angiopoietin-2, Flk-1 (vascular endothelial growth factor receptor-2), and vascular endothelial cadherin. The expression of PECAM-1 in tibial marrow cells was reduced at day 3 of TS to 80% and still showed significantly low levels at day 7 of TS to 72% of that at the respective days of GC. This decreased expression of PECAM-1 after 7-day TS showed the GC level at 5-day reloading after 7-day TS. However, the expression of PECAM-1 in humeral marrow cells (internal bone marrow control) after TS and RL remained unchanged and equivalent to that of GC. The expression level of PECAM-1 mRNA was significantly lower at day 7 of TS to 62% of that in GC. Double labeling analyses revealed that PECAM-1+ cells mostly consisted of endothelial cells and partially of granulocytes. In bone marrow cell cultures, the formation of alkaline phosphatase (ALP)+ colony forming units-fibroblastic was significantly reduced in the presence of anti-PECAM-1 antibody in the medium compared with the presence of immunoglobulin G (0.025 times as much as ALP production with immunoglobulin G). ALP production by cultured MC3T3-E1 was enhanced in combination with PECAM-1+ EOMA (1.8 times as much as ALP production by MC3T3-E1 alone), but not in combination with PECAM-1- ISOS-1. Anti-PECAM-1 antibody inhibited the increase in ALP production under co-culture with EOMA. CONCLUSIONS: Our data show that the reduced ALP activity after skeletal unloading is closely correlated with reduced expression of PECAM-1 in bone marrow cells. We speculate that the loss of osteogenic potential after skeletal unloading is caused by the suppression of PECAM-1 signaling on endothelial cellular surface.

Osteoclast development in immobilized bone is suppressed by parathyroidectomy in mice.

We tested the hypothesis that signaling of parathyroid hormone (PTH) facilitates osteoclastogenesis in bone marrow cells after immobilization, thereby reducing trabecular bone volume. We performed histomorphometric analyses in immobilized limbs after right sciatic neurectomy (IM) and in the contralateral limbs after sham surgery (M). Mice underwent thyroparathyroidectomy (TPTX) and then 0.2 microg/body of thyroxine was given three times a week, or the mice were subjected to sham surgery (sham). Six-week-old male ddY mice were assigned to four groups, as follows, after acclimatization for 1 week: M + sham, IM + sham; M + TPTX, and IM + TPTX. Bilateral tibial samples were used for analysis. Trabecular bone volume (BV/TV) in the secondary spongiosa of the proximal tibias in IM + sham was significantly reduced compared to that in M + sham. Osteoclast surface (Oc.S/BS) and number (Oc.N/BS) in IM + sham transiently increased at 3 and 4 weeks after IM. In contrast, TPTX partially prevented the IM-related reduction of BV/TV and completely suppressed the transient increases of Oc.S/BS and Oc.N/BS. In the bone marrow cells, the mRNA expression of RANKL was elevated in IM + sham, but not in IM + TPTX, compared to that in M + sham. The percentage of Mac-1-positive bone marrow cells, osteoclast precursors, was not altered after IM. There were no significant differences in the concentrations of interleukin (IL)-1alpha in the tibial bone marrow cell culture medium between M + sham and IM + sham. Our data demonstrated that significant increases in osteoclast surface and number after IM were suppressed in TPTX mice, closely associated with a reduction in the high expression of RANKL mRNA in the tibial bone marrow cells. We speculate that enhanced osteoclastogenesis due to limb immobilization may be related to the elevation of RANKL expression by the facilitation of parathyroid hormone signaling in bone marrow cells.