TGFβ1 Inhibits Migration, Invasion, and Proliferation of HTR-8/Svneo Cells via tTG-Activated TLR4/NF-κB Signaling Pathway.

TGFβ1 plays a key role in placental development, but its regulation of trophoblast function isn't fully known. Meanwhile, the TLR4/NF-κB pathway is linked to trophoblast dysfunction, yet the mediator linking TGFβ1 to this pathway is unclear. Thus, this study examines if tTG mediates TGFβ1's regulation of trophoblast functions via TLR4/NF-κB and its relevance in preeclampsia (PE). To address this, HTR-8/Svneo cells were treated with TGFβ1, cell migration, invasion, and proliferation were assessed via Transwell and CCK-8 assays. The expression of tTG, TLR4/NF-κB pathway components, and pro-inflammatory cytokines was evaluated by qPCR and Western blot. Gain- and loss-of-function approaches for tTG and TLR4 were employed, alongside transcriptome sequencing. The role of tTG's enzymatic activity was tested using the inhibitor Z-DON. Placental tissues from normal and preeclamptic pregnancies were analyzed for clinical validation. The results showed that TGFβ1 dose-dependently inhibited trophoblast migration, invasion, and proliferation while upregulating tTG expression. tTG overexpression activated the TLR4/NF-κB pathway and altered the transcriptome. TLR4 inhibition blocked tTG-mediated NF-κB activation. Furthermore, tTG knockdown and inhibiting tTG's enzymatic activity reversed the suppressive effects of TGFβ1 on cell functions and downstream TLR4/NF-κB signaling. Clinically, the protein levels of tTG and activated TLR4/NF-κB pathway components were significantly elevated in preeclamptic placentas. In conclusion, this study first reveals a TGFβ1/tTG/TLR4/NF-κB axis in trophoblasts, showing TGFβ1 inhibits trophoblast function via upregulation of tTG, which functionally leads to activation of the TLR4/NF-κB pathway in an enzyme activity-dependent manner, providing new insights into PE pathogenesis and potential therapeutic targets.