FIGURE 5.

HEY2 overexpression in osteoblasts from male mice induces resorption by co-cultured splenocytes and IL6 expression in vitro. Osteoblast-enriched cells were harvested from calvariae of male or female Col3.6-Hey2 transgenics (Hey2, black bars), or littermate wild type controls of the same sex (Wild Type, white bars) and cultured under conditions favoring osteoblastogenesis. In A, osteoblast-enriched cells from male mice were co-cultured with splenocytes from wild type mice of the same sex in the presence of 10 nm 1,25-dihydroxyvitamin D₃ (Vit.D) or vehicle. After 7 days, cells were removed with bleach, the culture substrate was stained by Von Kossa, and digital pictures were acquired for the estimation of the resorbed area. Data are expressed as the percentage of the resorbed area. Values are means ± S.E., n = 4–6. *, significantly different between HEY2 and wild type, p < 0.05. +, significantly different between 1,25-dihydroxyvitamin D₃ and vehicle, p < 0.05. In B, total RNA was extracted from osteoblast-enriched cell cultures at confluence, and mRNA was reverse-transcribed and amplified by qRT-PCR in the presence of specific primers. Data are expressed as Il6, Rankl, and Opg copy number, corrected for Gapdh copy number. Values are means ± S.E., n = 4. *, significantly different between HEY2 and wild type, p < 0.05.