Fig. 7.

Loss of Kalirin affects osteoclast and osteoblast activity. (A) Osteoclasts from Kal-KO and WT mice were differentiated with M-CSF (20 ng/ml) and RANKL (40 or 80 ng/ml) for 7 days, and TRAP-positive multinucleated osteoclasts were enumerated. (B) Osteoclasts were differentiated with RANKL and M-CSF and when mature (containing 3 or more nuclei) were replated onto dentin slices for 48 h. The area resorbed was stained with toluidine blue and measured using Bioquant software. The total area resorbed per dentin slice (n = 4/group) was normalized for the number of TRAP-positive osteoclasts. p < 0.05. (C) Mature osteoclasts were plated onto glass coverslips and stained with rhodamine phalloidin to detect actin. Nuclei were stained with DAPI. Scale bar is 10 μm. All experiments were reproduced 3–5 times with similar results and representative results are shown. (D) Equal numbers of calvarial osteoblasts (OB) were co-cultured with non-adherent bone marrow cells (BM) derived from either WT or Kal-KO mice as indicated. Cultures were differentiated with VitD₃ and PGE₂ for 6 days and the number of TRAP-positive osteoclasts was then enumerated. (E) Equal numbers of calvaria-derived osteoblastic cells from Kal-KO or WT neonates were differentiated in osteogenic media for 7 or 14 days. After 7 days, cultures were harvested and assayed for ALP activity. Results represent the average ± SEM for 10 independent experiments, expressed as a percentage relative to WT osteoblasts. (F) Stromal derived osteoblasts were differentiated in osteogenic media for 3–14 days and assayed for ALP (nmol/ml). Results show that loss of Kalirin increases ALP activity over time. (G) Fourteen day calvarial osteoblast cultures were examined for mineralizing activity using the Alizerin-S red elution assay. Results are the average ± SEM for four independent experiments. Results are expressed as a percentage relative to WT controls. (H) Stromal osteoblasts were differentiated for 7–21 days and assayed for mineralizing activity (Ca2+; nmol/ml). Samples were prepared in triplicate and averaged. Results reveal that loss of Kalirin inhibits the mineralizing activity of osteoblasts. Asterisks indicate p < 0.05 between WT and Kal-KO cells at the same day of differentiation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)