Lrp5 is not required for the proliferative response of osteoblasts to strain but regulates proliferation and apoptosis in a cell autonomous manner

Abstract

Although Lrp5 is known to be an important contributor to the mechanisms regulating bone mass, its precise role remains unclear. The aim of this study was to establish whether mutations in Lrp5 are associated with differences in the growth and/or apoptosis of osteoblast-like cells and their proliferative response to mechanical strain in vitro. Primary osteoblast-like cells were derived from cortical bone of adult mice lacking functional Lrp5 (Lrp5(-/-)), those heterozygous for the human G171V High Bone Mass (HBM) mutation (LRP5(G171V)) and their WT littermates (WT(Lrp5), WT(HBM)). Osteoblast proliferation over time was significantly higher in cultures of cells from LRP5(G171V) mice compared to their WT(HBM) littermates, and lower in Lrp5(-/-) cells. Cells from female LRP5(G171V) mice grew more rapidly than those from males, whereas cells from female Lrp5(-/-) mice grew more slowly than those from males. Apoptosis induced by serum withdrawal was significantly higher in cultures from Lrp5(-/-) mice than in those from WT(HBM) or LRP5(G171V) mice. Exposure to a single short period of dynamic mechanical strain was associated with a significant increase in cell number but this response was unaffected by genotype which also did not change the 'threshold' at which cells responded to strain. In conclusion, the data presented here suggest that Lrp5 loss and gain of function mutations result in cell-autonomous alterations in osteoblast proliferation and apoptosis but do not alter the proliferative response of osteoblasts to mechanical strain in vitro.

Figure 1. Proliferation of primary osteoblast-like cells derived from female and male LRP5 ^G171V and Lrp5 ^−/− mice and their WT littermates.

Osteoblast-like cells were cultured over 8 days and were fixed in absolute ice-cold MeOH on day 2, 4, 6 and 8. Cell's nuclei were stained using propidium iodide and counted using Microchip Type Automatic Cell Counter machine. Results are the mean ± SEM of three independent experiments. N = 4. Groups with the same letter are not significantly different. b vs. a  =  P<0.001. a + b vs. c  =  P<0.001.

Figure 2. Percentage of apoptosis in osteoblast-like cells 48 hours after treatment with 0.1% (A), 2.5% (B) and 10% (C) serum.

The TUNEL staining was used to determine the percentage of apoptotic cells in primary osteoblast-like cells derived from male and female WT_HBM, Lrp5 ^−/− and LRP5 ^G171V mice. Results are mean ± SEM of three independent experiments. Groups with the same letter are not significantly different.

Figure 3. Effects of 2500, 2900 and 3400 με on proliferation of primary osteoblast-like cells derived from female and male WT (A), Lrp5 ^−/− (B) and LRP5 ^G171V (C) mice.

Changes in absolute number of cells between static and strain of both genotypes and genders are shown. Results are mean ± SEM of three independent experiments. Experiments were repeated 3 times. No significant differences at 2500 and 2900 με were observed. ***p<0.001 and *p<0.05 compared with the static control within the gender. (D) The effects of 3400 με on proliferation of primary osteoblast-like cells derived from female and male LRP5 ^G171V and Lrp5 ^−/− mice and their WT littermates. Percentage differences between static and strain of both genotypes and genders are shown. Results are the mean ± SEM of three independent experiments. Experiments were repeated 3 times. There were no significant differences between groups.