The homeodomain protein TGIF plays crucial roles in tissue homeostasis. TGIF is phosphorylated in numerous cell systems, yet the responsible kinase(s) remains unidentified. In an effort to address this issue, we interrogated Eukaryotic Linear Motif (ELM) database, and found TGIF possesses a potential GSK3β phosphorylation site (T235 and T239). We then showed that GSK3β can directly phosphorylate TGIF, as demonstrated by in vitro kinase assays and western blotting using a specific antibody. Functionally, we found mutation of T235/T239 resulted in decreased TGIF turnover, providing an initial hint that phosphorylation by GSK3β might hinder TGIF stability. In fact, suppressing GSK3β activity through either genetic or chemical approaches triggered decreased TGIF polyubiquitination and clearance. Thus, similar to β-catenin, phosphorylation of TGIF by GSK3β?leads to its degradation. As GSK3β is a key kinase in Wnt signalling, we investigated whether TGIF played a physiological role in this pathway. Remarkably, expression of TGIF enhanced Wnt-induced gene expression, whereas TGIF deficiency elicited the opposite effects. Mechanistically, TGIF appeared to promote β-catenin accumulation, interfering with the assembly of the β-catenin destruction complex. Furthermore, activation of Wnt signalling induced the expression of TGIF itself, revealing an ability of TGIF to govern a feed-forward loop that sustains Wnt signalling. Given that Wnt signalling is a regulator of osteoblast differentiation and bone formation, we then tested whether TGIF was capable of enhancing this pathway in osteoblasts (OBs) and bone formation in vitro and in vivo. Expressing TGIF increased OB differentiation in the pre-OB cell lines, ST2 and C3H10T1/2 through Wnt signalling activation, as TGIF depletion was sufficient to blunt Wnt3a-induced osteoblast differentiation in these cells. In vivo, TGIF-/- mice display decreased osteoblast differentiation and low bone mass. More importantly, deletion of TGIF prevented the high bone mass phenotype seen in mice harbouring heterozygote deletion of DKK1. This study therefore establishes TGIF as a component of the Wnt signalling machinery that is required for efficient Wnt-induced osteoblast differentiation and bone formation.
Disclosure: The authors declared no competing interests. This work was supported by NIH RO1-AR48218 to RB and NIH RO1-AR059070 to AA.