Tgif1-deficiency impairs cytoskeletal architecture in osteoblasts by suppressing PAK3 signaling

Reviewer #1 (Public Review):

Summary:
The authors were trying to achieve that Tgif1 expression is regulated by EAK1/2 and PTH in a time-dependent manner, and its roles in suppressing Pak3 for facilitating osteoblast adhesion. The authors further tried to show that the Tgif1-Pak3 signaling plays a significant role in osteoblast migration to the site of bone repair and bone remodeling.

Strengths:
- In a previous study, it was demonstrated that Tgif1 is a target gene of PTH, and the absence of Tgif1 failed to increase bone mass by PTH treatment (Saito et al., Nat Commun., 2019). In this study, the authors found that Tgif1-Pak3 signaling prompts osteoblast migration through osteoblast adhesion to prompt bone regeneration. This novel finding provides a better understanding of how Tgif1 expression in osteoblasts regulates adherence, spreading, and migration during bone healing and bone remodeling.

- The authors demonstrated that ERK1/2 and PTH regulate Tgif1 expression in a time-dependent manner and its role in suppressing Pak3 through various experimental approaches such as luciferase assay, ChIP assay, and gene silencing. These results contribute to the overall strength of the article.

Weaknesses:
-The authors need to further justify why they focused on Pak3 in the introduction by mentioning its known function for cell adhesion.

-Some results indicated statistically significant but small changes. The authors need to explain in the discussion part why they believe this is the major mechanism or why there may be some other possible mechanisms.

-The study does not include enough in vivo data to claim that this mechanism is crucial for bone healing and bone remodeling in vivo.

Reviewer #2 (Public Review):

Summary:
Bolamperti S. et al. 2023 investigate whether the expression of TG-interacting factor (Tgif1) is essential for osteoblastic cellular activity regarding morphology, adherence, migration/recruitment, and repair. Towards this end, germ-line Tgif1 deletion (Tgif1-/-) mice or male mice lacking expression of Tgif1 in mature osteoblastic and osteocytic cells (Dmp1-Cre+; Tgif1fl/fl) and corresponding controls were studied in physiological, bone anabolic, and bone fracture-repair conditions. Both Tgif1-/- and Dmp1-Cre+; Tgif1fl/fl exhibited decreased osteoblasts on cancellous bone surfaces and adherent to collagen I-coated plates. Tgif1-/- mice exhibit impaired healing in the tibial midshaft fracture model, as indicated by decreased bone volume (BV/Cal.V), osteoid (OS/BS), and low osteoblasts (number and surface). Likewise, both Tgif1-/- and Dmp1-Cre+; Tgif1fl/fl show impaired PTH 1-34, (100 µg/kg, 5x/wk for 3 wks) osteoblast activation in vivo, as detected by increases in quiescent bone surfaces. Mechanistic in vitro studies then utilized primary osteoblasts isolated from Tgif1-/- mice and siRNA Tgif1 knockdown OCY454 cells to further investigate and identify the downstream Tgif1 target driving these osteoblastic impairments. In vitro, Tgif1-/- osteoblastic and Tgif1 knockdown OCY454 cells exhibit decreased migration, abnormal morphology, and decreased focal adhesions/cells. Unexpectantly though, localization assays revealed Tgif1 to primarily concentrate in the nucleus and not to co-localize with focal adhesions (paxillin, talin). Also, the expression of major focal adhesion components (paxillin, talin, FAK, Src, etc.) or the Cdc42 family was not altered by loss of Tgif1 expression. In contrast, PAK3 expression is markedly upregulated by loss of Tgif1. In silico analysis followed by mechanistic molecular assays involving ChIP, siRNA (Tgif1, PAK3), and transfection (rat PAK3 promoter) techniques show that Tgif1 physically binds to a specific site in the PAK3 promoter region. Further, the knockdown of PAK3 rescues the Tgif1-deficient abnormal morphology in OCY454 cells. This is the first study to identify the novel transcriptional repression of PAK3 by Tgif1 as well as the specific Tgif1 binding site within the PAK3 promoter.

Strengths:
This work has a plethora of strengths. The co-authors achieved their aim of eliciting the role of Tgif1 expression in osteoblastic cellular functions (morphology, spreading/attachment, migration). Further, this work is the first to depict the novel mechanism of Tgif1 transcriptional repression of PAK3 by a thorough usage of mechanistic molecular assays (in silico analysis, ChIP, siRNA, transfection etc.). The conclusions are well supported and justified by these findings, as the appropriate controls, sample sizes (statistical power), statistics, and assays were fully utilized.

The claims and conclusions are justified by the data.

Weaknesses:
The discussion section could be expanded with a few sentences regarding limitations to the current study and potential future directions.