Figure 8

TG2 deficiency augments the activation of the NF-κB signaling pathway by RANKL. (a) WT and TG2 KO BMMs were serum-starved for 5 h and stimulated with RANKL (500 ng/ml) for the indicated times. Whole cell lysates were analyzed by Western blot using anti-phospho-IκB and -IκB antibodies. (b) Nuclear and cytosolic fractions prepared from WT and TG2 KO BMMs were stimulated with RANKL and subjected to Western blot analysis. PARP (full length) and α-tubulin were used as loading controls for nuclear and cytoplasmic extracts, respectively. The ratios of p65 to PARP were determined by densitometry. (c) WT and TG2 KO BMMs were serum-starved for 5 h and stimulated with or without RANKL (500 ng/ml) for 15 min. Cells were stained for p65 and laminB. Anti-laminB staining marked the nuclear envelope. Cells with nuclear p65 staining were counted. Scale bar, 20 μm. *P < 0.05 versus WT cells. (d) Schematic illustration of a proposed mechanism through which TG2 regulates osteoclastogenesis. A reduction in TG2 enhances the activation of NF-κB p65, which increases the expression of Blimp1. Blimp1 together with c-Fos augments the expression and nuclear translocation of NFATc1 to enhance osteoclastogenesis induced by RANKL. Full length Western blots are presented in Supplementary Figure 10.