Figure 4.

Increased bone marrow adiposity and RANKL production in 3-month-old Tgm2^−/−;F13a1^−/− mice. (a) Histomorphometric quantification of osteoblasts (N.Ob/T.Ar) and osteocytes (N.Ocy/T.Ar) (relative to trabecular area) show no significant change in 3-month-old Tgm2^−/−;F13a1^−/− mice. n=6. (b) Calcein double-labeling and its quantification shows no change in mineral apposition rate (MAR). n=6. Images are representative of six mice. (c) ALP staining (pink) shows active osteoblasts around trabeculae and reveals increased adipocity of the bone marrow (black arrow heads pointing to adipocytes that appear in white) in the in Tgm2^−/−;F13a1^−/− mice. Images are representative of three mice. (d) Quantification of adipocyte number in serial sections of bone marrow confirms the significant increase. n=3. (e and f) bmMSCs isolated from WT and Tgm2^−/−;F13a1^−/− mouse bone marrow show increased potential for adipogenesis under adipogenic conditions as assessed and quantified by Oil Red staining at day 13 of culture. Images are representative of three separate experiments. n=3 in Oil Red quantification. (g). RT-PCR analysis of bmMSC gene expression. The adipocyte marker, Pparg, is increased in Tgm2^−/−;F13a1^−/− cells grown in adipogenic media. Rankl production is dramatically increased in TG2 and FXIII-A deficient bmMSCs. Conversely Opg production is decreased. Csf1 production does not show change in the absence of the two TGs. Representative data from three separate experiments. P-values are as follows: *P<0.05, ***P<0.001, NS; not significant. Scale bar, 20 μm (b); and 200 μm (c)