A pleiotropic immunoregulatory cytokine, TGF-β, signals via the receptor-regulated SMADs: SMAD2 and SMAD3, which are constitutively expressed in normal cells. Here, we show that selective repression of SMAD3 induces cDC differentiation from the CD115+ common DC progenitor (CDP). SMAD3 was expressed in haematopoietic cells including the macrophage DC progenitor. However, SMAD3 was specifically down-regulated in CD115+ CDPs, SiglecH- pre-DCs, and cDCs, whereas SMAD2 remained constitutive. SMAD3-deficient mice showed a significant increase in cDCs, SiglecH- pre-DCs, and CD115+ CDPs compared with the littermate control. SMAD3 repressed the mRNA expression of FLT3 and the cDC-related genes: IRF4 and ID2. We found that one of the SMAD transcriptional corepressors, c-SKI, cooperated with phosphorylated STAT3 at Y705 and S727 to repress the transcription of SMAD3 to induce cDC differentiation. These data indicate that STAT3 and c-Ski induce cDC differentiation by repressing SMAD3: the repressor of the cDC-related genes during the developmental stage between the macrophage DC progenitor and CD115+ CDP.

Glucose-regulated protein 78 (GRP78), also termed HSPA5, was widely studied in cancer. It was recently approved that GRP78 has nuclear localization potential that sheds light on its role in cancer development. The inhibitor of DNA binding and differentiation 2 (ID2) is the nuclear component that associates with GRP78. The interaction between these two proteins is not understood clearly. In the current study, the binding pattern of GRP78/ID2 is predicted using computational methods. Protein-protein docking is used along with molecular dynamics simulation. The substrate binding domain β of GRP78 can stably interact with the loop region (C42-S60) of ID2 as predicted in this study. This paves the way for a possible destabilizer for this association and cancer eradication.

Cytoplasmic polyadenylation element-binding protein 4 (Cpeb4) is an RNA-binding protein that regulates posttranscriptional regulation, such as regulation of messenger RNA stability and translation. In the previous study, we reported that Cpeb4 localizes to nuclear bodies upon induction of osteoclast differentiation by RANKL. However, the mechanisms of the localization of Cpeb4 and osteoclastogenesis by Cpeb4 remain unknown. Here, we show that Cpeb4 localizes to the nuclear bodies by its RNA-binding ability and partially regulates normal splicing during osteoclast differentiation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with Phos-tag® revealed that the phosphorylation levels of Cpeb4 were already high in the RAW264.7 cells and were not altered by RANKL treatment. Immunofluorescence showed that exogenous Cpeb4 in HEK293T cells without RANKL stimulation localized to the same foci as shown in RANKL-stimulated RAW264.7 cells. Furthermore, when nuclear export was inhibited by leptomycin B treatment, Cpeb4 accumulated throughout the nucleus. Importantly, RNA recognition motif (RRM) 7 of Cpeb4 was essential for the localization. In contrast, the intrinsically disordered region, RRM1, and zinc finger domain CEBP_ZZ were not necessary for the localization. The mechanistic study showed that Cpeb4 co-localized and interacted with the splicing factors serine/arginine-rich splicing factor 5 (SRSF5) and SRSF6, suggesting that Cpeb4 may be involved in the splicing reaction. RNA-sequencing analysis revealed that the expression of genes related to cell proliferation processes, such as mitotic cell cycle and regulation of cell cycle processes, was elevated in osteoclasts depleted of Cpeb4. Interestingly, the splicing pattern of the inhibitor of DNA binding 2 (Id2) gene, which suppresses osteoclast differentiation, was altered by the depletion of Cpeb4. These results provide new insight into the role of Cpeb4 as a player of normal splicing of Id2 in osteoclast differentiation.

BACKGROUND: Inhibitor of DNA Binding 2 (ID2) plays a crucial role in tumor cell proliferation, invasion, metastasis, and stemness. Aberrant ID2 expression is associated with poor prognosis in various cancers. However, the specific function of ID2 in thyroid cancer remain unclear.
METHODS: The TCGA database were utilized to explore the clinical relevance of ID2 in cancer. GO, KEGG, and TIMER were employed to predict the potential roles of ID2 in cancer. Functional analysis, including CCK-8, colony formation, transwell, wound healing, and sphere formation experiments, were conducted to determine the biological functions of ID2 in human cancers. Western blot (WB), RT-qPCR, and immunohistochemical (IHC) analyses were used to investigate the relationship between ID2 and downstream targets.
RESULTS: Our study revealed significant overexpression of ID2 in various malignant tumor cells. Knocking ID2 significantly inhibited cancer cell proliferation and invasion, while overexpressing ID2 enhanced these capabilities. Additionally, ID2 mediates resistance of cancer cells to protein kinase B (or Akt) inhibitions. Further WB and IHC experiments indicated that ID2 promotes the phosphorylation activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, thereby upregulating the expression of downstream proliferation, epithelial-mesenchymal transition (EMT), and stemness-related markers.
CONCLUSIONS: We found that ID2 significantly promotes thyroid cancer cell proliferation, migration, EMT, and stemness through the PI3K/Akt pathway. Moreover, ID2 plays a crucial role in regulating cancer immune responses. It may serve as a potential biomarker for enhancing the efficacy of chemotherapy, targeted therapy, and immunotherapy against cancer.

BACKGROUND: Inhibitors of DNA binding (ID) proteins mainly inhibit gene expression and regulate cell fate decisions by interacting with E-proteins. All four ID proteins (ID1-4) are present in the testis, and ID4 has a particularly important role in spermatogonial stem cell fate determination. Several lines of evidence indicate that ID proteins are involved in meiosis; however, functional experiments have not been conducted to validate this observation.
RESULTS: In this study, we report that ID2 is enriched in spermatocytes and that forced ID2 expression in germ cells causes defects in spermatogenesis. A detailed analysis demonstrated that Id2 overexpression (Id2 OE) decreased the total number of spermatogonia and changed the dynamics of meiosis progression. Specifically, spermatocytes were enriched in the zygotene stage, and the proportion of pachytene spermatocytes was significantly decreased, indicating defects in the zygotene-pachytene transition. The number of MLH1-positive foci per cell was decreased in pachytene spermatocytes from Id2 OE testes, suggesting abnormalities in recombination. Transcriptome analysis revealed that forced Id2 expression changed the expression of a list of genes mainly associated with meiosis and spermatid development.
CONCLUSIONS: ID2 protein is expressed in spermatocytes, and its genetic ablation in the germline does not affect spermatogenesis, likely due to genetic compensation of its family members. However, forced Id2 expression changes meiosis progression and causes defects in spermiogenesis. These data provide important evidence that ID proteins play pivotal roles in male meiosis and spermatid development.

暂无摘要。

In eukaryotes, N6-methyladenosine (m6A) is the most prevalent epigenetic alteration. Methyltransferase-like 3 (METTL3) is a key player in the control of m6A, although its function in pancreatic cancer is incompletely understood. In this study, we examined the role that METTL3 plays in pancreatic cancer cell proliferation and stemness. We discovered that in pancreatic cancer cells, METTL3-mediated m6A alterations regulate ID2 as a downstream target. The stability of ID2 mRNA was decreased and m6A modification was effectively eliminated by METTL3 knockdown in pancreatic cancer cells. We also demonstrate that m6a-YTHDF2 is necessary for the METTL3-mediated stabilization of ID2 mRNA. Additionally, we show that ID2 controls the stemness molecules NANOG and SOX2 via the PI3K-AKT pathway to support pancreatic cancer growth and stemness maintenance. Our data suggest that METTL3 may post-transcriptionally upregulate ID2 expression in an m6A-YTHDF2-dependent manner to further promote the stabilization of ID2 mRNA, which may be a new target for pancreatic cancer treatment.

Obesity is a risk factor for preeclampsia. We investigated how obesity influences preeclampsia in mice lacking ankyrin-repeat-and-SOCS-box-containing-protein 4 (ASB4), which promotes trophoblast differentiation via degrading the inhibitor of DNA-binding protein 2 (ID2). Asb4-/- mice on normal chow (NC) develop mild preeclampsia-like phenotypes during pregnancy, including hypertension, proteinuria, and reduced litter size. Wild-type (WT) and Asb4-/- females were placed on a high-fat diet (HFD) starting at weaning. At the age of 8-9 weeks, they were mated with WT or Asb4-/- males, and preeclamptic phenotypes were assessed. HFD-WT dams had no obvious adverse outcomes of pregnancy. In contrast, HFD-Asb4-/- dams had significantly more severe preeclampsia-like phenotypes compared to NC-Asb4-/- dams. The HFD increased white fat weights and plasma leptin and insulin levels in Asb4-/- females. In the HFD-Asb4-/- placenta, ID2 amounts doubled without changing the transcript levels, indicating that insulin likely increases ID2 at a level of post-transcription. In human first-trimester trophoblast HTR8/SVneo cells, exposure to insulin, but not to leptin, led to a significant increase in ID2. HFD-induced obesity markedly worsens the preeclampsia-like phenotypes in the absence of ASB4. Our data indicate that hyperinsulinemia perturbs the timely removal of ID2 and interferes with proper trophoblast differentiation, contributing to enhanced preeclampsia.

Activin A (Act A) has been reported to promote oligodendrocyte progenitor cell (OPC) differentiation in vitro and improve neurological outcomes in adult mice. However, the roles and mechanisms of action of Act A in preterm brain injury are unknown. In the present study, P5 rats were subjected to hypoxia‑ischemia to establish a neonatal white matter injury (WMI) model and Act A was injected via the lateral ventricle. Pathological characteristics, OPC differentiation, myelination, and neurological performance were analyzed. Further, the involvement of the Noggin/BMP4/Id2 signaling pathway in the roles of Act A in WMI was explored. Act A attenuated pathological damage, promoted OPC differentiation, enhanced myelin sheath and myelinated axon formation, and improved neurological performance of WMI rats. Moreover, Act A enhanced noggin expression, which, in turn, inhibited the expression of bone morphogenetic protein 4 (BMP4) and inhibitor of DNA binding 2 (Id2). Furthermore, upregulation of Id2 completely abolished the rescue effects of Act A in WMI rats. In conclusion, the present findings suggested that Act A rescues preterm brain injury via targeting a novel Noggin/BMP4/Id2 signaling pathway.

Components of bone morphogenetic protein (BMP) signalling have been implicated in both pathogenesis of pulmonary arterial hypertension (PAH) and endothelial-mesenchymal transition (EndoMT). In particular, the importance of BMP type 2 receptor in these processes has been extensively analysed. However, the contribution of BMP type 1 receptors (BMPR1s) to the onset of PAH and EndoMT remains poorly understood. BMPR1A, one of BMPR1s, was recently implicated in the pathogenesis of PAH, and was found to be down-regulated in the lungs of PAH patients, neither the downstream mechanism nor its contribution to EndoMT has been described. Therefore, we aim to delineate the role of endothelial BMPR1A in modulating EndoMT and pathogenesis of PAH.
We find that BMPR1A knockdown in endothelial cells (ECs) induces hallmarks of EndoMT, and deletion of endothelial Bmpr1a in adult mice (Bmpr1aiECKO) leads to development of PAH-like symptoms due to excessive EndoMT. By lineage tracing, we show that endothelial-derived smooth muscle cells are increased in endothelial Bmpr1a-deleted mice. Mechanistically, we identify ZEB1 as a primary target for BMPR1A in this setting; upon BMPR1A activation, ID2 physically interacts and sequesters ZEB1 to attenuate transcription of Tgfbr2, which in turn lowers the responses of ECs towards transforming growth factor beta (TGFβ) stimulation and prevents excessive EndoMT. In Bmpr1aiECKO mice, administering endothelial targeting lipid nanoparticles containing siRNA against Tgfbr2 effectively ameliorate PAH, reiterating the importance of BMPR1A-ID2/ZEB1-TGFBR2 axis in modulating progression of EndoMT and pathogenesis of PAH.
We demonstrate that BMPR1A is key to maintain endothelial identity and to prevent excessive EndoMT. We identify BMPR1A-induced interaction between ID2 and ZEB1 is the key regulatory step for onset of EndoMT and pathogenesis of PAH. Our findings indicate that BMPR1A-ID2/ZEB1-TGFBR2 signalling axis could serve as a potential novel therapeutic target for PAH and other EndoMT-related vascular disorders.