HMGB1 regulates autophagy of placental trophoblast through ERK signaling pathway.

OBJECTIVE: The purpose of this study is to investigate the role of high mobility group protein B1 (HMGB1) in placental development and fetal growth. METHODS: We employed the Cre-loxP recombination system to establish a placenta-specific HMGB1 knockout mouse model. Breeding HMGB1flox/flox mice with Elf5-Cre mice facilitated the knockout, leveraging Elf5 expression in extra-embryonic ectoderm, ectoplacental cone, and trophoblast giant cells at 12.5 days of embryonic development. The primary goal of this model was to elucidate the molecular mechanism of HMGB1 in placental development, assessing parameters such as placental weight, fetal weight, and bone development. Additionally, we utilized lentiviral interference and overexpression of HMGB1 in human trophoblast cells to further investigate HMGB1's functional role. RESULTS: Our findings indicate that HMGB1flox/floxElf5cre/+ mouse display fetal growth restriction (FGR), characterized by decreased placental and fetal weight and impaired bone development. And the absence of HMGB1 inhibits autophagosome formation, impairs lysosomal degradation, and disrupts autophagic flux. Depletion of HMGB1 in human trophoblast cells also suppresses cell viability, proliferation, migration, and invasion by inhibiting the ERK signaling pathway. Overexpression of HMGB1 observed the opposite phenotypes. CONCLUSIONS: HMGB1 participates in the regulation of autophagy through the ERK signaling pathway and affects placental development.

TBX3 reciprocally controls key trophoblast lineage decisions in villi during human placenta development in the first trimester.

Human trophoblastic lineage development is intertwined with placental development and pregnancy outcomes, but the regulatory mechanisms underpinning this process remain inadequately understood. In this study, based on single-nuclei RNA sequencing (snRNA-seq) analysis of the human early maternal-fetal interface, we compared the gene expression pattern of trophoblast at different developmental stages. Our findings reveal a predominant upregulation of TBX3 during the transition from villous cytotrophoblast (VCT) to syncytiotrophoblast (SCT), but downregulation of TBX3 as VCT progresses into extravillous trophoblast cells (EVT). Immunofluorescence analysis verified the primary expression of TBX3 in SCT, partial expression in MKi67-positive VCT, and absence in HLA-G-positive EVT, consistent with our snRNA-seq results. Using immortalized trophoblastic cell lines (BeWo and HTR8/SVneo) and human primary trophoblast stem cells (hTSCs), we observed that TBX3 knockdown impedes SCT formation through RAS-MAPK signaling, while TBX3 overexpression disrupts the cytoskeleton structure of EVT and hinders EVT differentiation by suppressing FAK signaling. In conclusion, our study suggests that the spatiotemporal expression of TBX3 plays a critical role in regulating trophoblastic lineage development via distinct signaling pathways. This underscores TBX3 as a key determinant during hemochorial placental development.

Appropriate expression of P57kip2 drives trophoblast fusion via cell cycle arrest.

The syncytiotrophoblast, derived from cytotrophoblast fusion, is responsible for maternal-fetal exchanges, secretion of pregnancy-related hormones, and fetal defense against pathogens. Inadequate cytotrophoblast fusion can lead to pregnancy disorders, such as preeclampsia and fetal growth restriction. However, little is known about the mechanism of cytotrophoblast fusion in both physiological and pathological pregnancy conditions. In this study, P57kip2 (P57), a cell cycle-dependent kinase inhibitor that negatively regulates the cell cycle, was found to be up-regulated during the process of syncytialization in both primary trophoblast cells and BeWo cells. Co-immunofluorescence with proliferation markers Ki67 and Cyclin-CDK factors further showed that P57 specifically localizes in the post-mitotic cytotrophoblast subtype of the early pregnancy villi. Overexpression of P57 promoted trophoblast syncytialization by arresting the cell cycle at the G1/G0 phase and inhibiting proliferation. Blocking of the cell cycle through a serum starvation culture resulted in an enhancement of cytotrophoblast fusion and the up-regulation of P57. In both spontaneous cytotrophoblast fusion and forskolin-induced BeWo cell fusion models, an initial up-regulation of P57 was observed followed by a subsequent down-regulation. These findings indicate that proper expression of P57 at cytotrophoblast differentiation nodes plays an important role in trophoblast syncytialization.