Endothelial Cell Flow-Mediated Quiescence Is Temporally Regulated and Utilizes the Cell Cycle Inhibitor p27.

BACKGROUND: Endothelial cells regulate their cell cycle as blood vessels remodel and transition to quiescence downstream of blood flow-induced mechanotransduction. Laminar blood flow leads to quiescence, but how flow-mediated quiescence is established and maintained is poorly understood. METHODS: Primary human endothelial cells were exposed to laminar flow regimens and gene expression manipulations, and quiescence depth was analyzed via time-to-cell cycle reentry after flow cessation. Mouse and zebrafish endothelial expression patterns were examined via scRNA-seq (single-cell RNA sequencing) analysis, and mutant or morphant fish lacking p27 were analyzed for endothelial cell cycle regulation and in vivo cellular behaviors. RESULTS: Arterial flow-exposed endothelial cells had a distinct transcriptome, and they first entered a deep quiescence, then transitioned to shallow quiescence under homeostatic maintenance conditions. In contrast, venous flow-exposed endothelial cells entered deep quiescence early that did not change with homeostasis. The cell cycle inhibitor p27 (CDKN1B) was required to establish endothelial flow-mediated quiescence, and expression levels positively correlated with quiescence depth. p27 loss in vivo led to endothelial cell cycle upregulation and ectopic sprouting, consistent with loss of quiescence. HES1 and ID3, transcriptional repressors of p27 upregulated by arterial flow, were required for quiescence depth changes and the reduced p27 levels associated with shallow quiescence. CONCLUSIONS: Endothelial cell flow-mediated quiescence has unique properties and temporal regulation of quiescence depth that depends on the flow stimulus. These findings are consistent with a model whereby flow-mediated endothelial cell quiescence depth is temporally regulated downstream of p27 transcriptional regulation by HES1 and ID3. The findings are important in understanding endothelial cell quiescence misregulation that leads to vascular dysfunction and disease.

ID3 is a novel target gene of p53 and modulates lung cancer cell metastasis.

The tumor suppressor p53 prevents cancer development by regulating dozens of target genes with diverse biological functions. Although numerous p53 target genes have been identified to date, the dynamics and function of the regulatory network centered on p53 have not yet been fully elucidated. We herein identified inhibitor of DNA-binding/differentiation-3 (ID3) as a direct p53 target gene. p53 bound the distal promoter of ID3 and positively regulated its transcription. ID3 expression was significantly decreased in clinical lung cancer tissues, and was closely associated with overall survival outcomes in these patients. Functionally, ID3 deficiency promoted the metastatic ability of lung cancer cells through its effects on the transcriptional regulation of CDH1. Furthermore, the ectopic expression of ID3 in p53-knockdown cells restored E-cadherin expression. Collectively, the present results demonstrate that ID3 plays a tumor-suppressive role as a downstream effector of p53 and impedes lung cancer cell metastasis by regulating E-cadherin expression.

Silibinin is a suppressor of the metastasis-promoting transcription factor ID3.

BACKGROUND: ID3 (inhibitor of DNA binding/differentiation-3) is a transcription factor that enables metastasis by promoting stem cell-like properties in endothelial and tumor cells. The milk thistle flavonolignan silibinin is a phytochemical with anti-metastatic potential through largely unknown mechanisms. HYPOTHESIS/PURPOSE: We have mechanistically investigated the ability of silibinin to inhibit the aberrant activation of ID3 in brain endothelium and non-small cell lung cancer (NSCLC) models. METHODS: Bioinformatic analyses were performed to investigate the co-expression correlation between ID3 and bone morphogenic protein (BMP) ligands/BMP receptors (BMPRs) genes in NSCLC patient datasets. ID3 expression was assessed by immunoblotting and qRT-PCR. Luciferase reporter assays were used to evaluate the gene sequences targeted by silibinin to regulate ID3 transcription. In silico computational modeling and LanthaScreen TR-FRET kinase assays were used to characterize and validate the BMPR inhibitory activity of silibinin. Tumor tissues from NSCLC xenograft models treated with oral silibinin were used to evaluate the in vivo anti-ID3 effects of silibinin. RESULTS: Analysis of lung cancer patient datasets revealed a top-ranked positive association of ID3 with the BMP9 endothelial receptor ACVRL1/ALK1 and the BMP ligand BMP6. Silibinin treatment blocked the BMP9-induced activation of the ALK1-phospho-SMAD1/5-ID3 axis in brain endothelial cells. Constitutive, acquired, and adaptive expression of ID3 in NSCLC cells were all significantly downregulated in response to silibinin. Silibinin blocked ID3 transcription via BMP-responsive elements in ID3 gene enhancers. Silibinin inhibited the kinase activities of BMPRs in the micromolar range, with the lower IC50 values occurring against ACVRL1/ALK1 and BMPR2. In an in vivo NSCLC xenograft model, tumoral overexpression of ID3 was completely suppressed by systematically achievable oral doses of silibinin. CONCLUSIONS: ID3 is a largely undruggable metastasis-promoting transcription factor. Silibinin is a novel suppressor of ID3 that may be explored as a novel therapeutic approach to interfere with the metastatic dissemination capacity of NSCLC.

ID factors regulate the ability of Müller glia to become proliferating neurogenic progenitor-like cells.

The purpose of this study was to investigate how ID factors regulate the ability of Müller glia (MG) to reprogram into proliferating MG-derived progenitor cells (MGPCs) in the chick retina. We found that ID1 is transiently expressed by maturing MG (mMG), whereas ID4 is maintained in mMG in embryonic retinas. In mature retinas, ID4 was prominently expressed by resting MG, but following retinal damage ID4 was rapidly upregulated and then downregulated in MGPCs. By contrast, ID1, ID2, and ID3 were low in resting MG and then upregulated in MGPCs. Inhibition of ID factors following retinal damage decreased numbers of proliferating MGPCs. Inhibition of IDs, after MGPC proliferation, significantly increased numbers of progeny that differentiated as neurons. In damaged or undamaged retinas inhibition of IDs increased levels of p21Cip1 in MG. In response to damage or insulin+FGF2 levels of CDKN1A message and p21Cip1 protein were decreased, absent in proliferating MGPCs, and elevated in MG returning to a resting phenotype. Inhibition of notch- or gp130/Jak/Stat-signaling in damaged retinas increased levels of ID4 but not p21Cip1 in MG. Although ID4 is the predominant isoform expressed by MG in the chick retina, id1 and id2a are predominantly expressed by resting MG and downregulated in activated MG and MGPCs in zebrafish retinas. We conclude that ID factors have a significant impact on regulating the responses of MG to retinal damage, controlling the ability of MG to proliferate by regulating levels of p21Cip1, and suppressing the neurogenic potential of MGPCs.

The nuclear factor ID3 endows macrophages with a potent anti-tumour activity.

Macrophage activation is controlled by a balance between activating and inhibitory receptors1-7, which protect normal tissues from excessive damage during infection8,9 but promote tumour growth and metastasis in cancer7,10. Here we report that the Kupffer cell lineage-determining factor ID3 controls this balance and selectively endows Kupffer cells with the ability to phagocytose live tumour cells and orchestrate the recruitment, proliferation and activation of natural killer and CD8 T lymphoid effector cells in the liver to restrict the growth of a variety of tumours. ID3 shifts the macrophage inhibitory/activating receptor balance to promote the phagocytic and lymphoid response, at least in part by buffering the binding of the transcription factors ELK1 and E2A at the SIRPA locus. Furthermore, loss- and gain-of-function experiments demonstrate that ID3 is sufficient to confer this potent anti-tumour activity to mouse bone-marrow-derived macrophages and human induced pluripotent stem-cell-derived macrophages. Expression of ID3 is therefore necessary and sufficient to endow macrophages with the ability to form an efficient anti-tumour niche, which could be harnessed for cell therapy in cancer.

ID4-dependent secretion of VEGFA enhances the invasion capability of breast cancer cells and activates YAP/TAZ via integrin ß3-VEGFR2 interaction.

Understanding the mechanisms of breast cancer cell communication underlying cell spreading and metastasis formation is fundamental for developing new therapies. ID4 is a proto-oncogene overexpressed in the basal-like subtype of triple-negative breast cancer (TNBC), where it promotes angiogenesis, cancer stem cells, and BRACA1 misfunction. Here, we show that ID4 expression in BC cells correlates with the activation of motility pathways and promotes the production of VEGFA, which stimulates the interaction of VEGFR2 and integrin ß3 in a paracrine fashion. This interaction induces the downstream focal adhesion pathway favoring migration, invasion, and stress fiber formation. Furthermore, ID4/ VEGFA/ VEGFR2/ integrin ß3 signaling stimulates the nuclear translocation and activation of the Hippo pathway member's YAP and TAZ, two critical executors for cancer initiation and progression. Our study provides new insights into the oncogenic roles of ID4 in tumor cell migration and YAP/TAZ pathway activation, suggesting VEGFA/ VEGFR2/ integrin ß3 axis as a potential target for BC treatment.

ALDOA coordinates PDE3A through the ß-catenin/ID3 axis to stimulate cancer metastasis and M2 polarization in lung cancer with EGFR mutations.

Lung cancer has a high incidence rate and requires more effective treatment strategies and drug options for clinical patients. EGFR is a common genetic alteration event in lung cancer that affects patient survival and drug strategy. Our study discovered aberrant aldolase A (ALDOA) expression and dysfunction in lung cancer patients with EGFR mutations. In addition to investigating relevant metabolic processes like glucose uptake, lactate production, and ATPase activity, we examined multi-omics profiles (transcriptomics, proteomics, and pull-down assays). It was observed that phosphodiesterase 3A (PDE3A) enzyme and ALDOA exhibit correlation, and furthermore, they impact M2 macrophage polarization through ß-catenin and downstream ID3. In addition to demonstrating the aforementioned mechanism of action, our experiments discovered that the PDE3 inhibitor trequinsin has a substantial impact on lung cancer cell lines with EGFR mutants. The trequinsin medication was found to decrease the M2 macrophage polarization status and several cancer phenotypes, in addition to transduction. These findings have potential prognostic and therapeutic applications for clinical patients with EGFR mutation and lung cancer.

Inhibitor of DNA Binding Protein 3 (ID3) and Nuclear Respiratory Factor 1 (NRF1) Mediated Transcriptional Gene Signatures are Associated with the Severity of Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA) is a degenerative vasculopathy. We have previously shown that transcription regulating proteins- inhibitor of DNA binding protein 3 (ID3) and the nuclear respiratory factor 1 (NRF1) contribute to vascular dysregulation. In this study, we have identified sex specific ID3 and NRF1-mediated gene networks in CAA patients diagnosed with Alzheimer's Disease (AD). High expression of ID3 mRNA coupled with low NRF1 mRNA levels was observed in the temporal cortex of men and women CAA patients. Low NRF1 mRNA expression in the temporal cortex was found in men with severe CAA. High ID3 expression was found in women with the genetic risk factor APOE4. Low NRF1 expression was also associated with APOE4 in women with CAA. Genome wide transcriptional activity of both ID3 and NRF1 paralleled their mRNA expression levels. Sex specific differences in transcriptional gene signatures of both ID3 and NRF1 were observed. These findings were further corroborated by Bayesian machine learning and the GeNIe simulation models. Dynamic machine learning using a Monte Carlo Markov Chain (MCMC) gene ordering approach revealed that ID3 was associated with disease severity in women. NRF1 was associated with CAA and severity of this disease in men. These findings suggest that aberrant ID3 and NRF1 activity presumably plays a major role in the pathogenesis and severity of CAA. Further analyses of ID3- and NRF1-regulated molecular drivers of CAA may provide new targets for personalized medicine and/or prevention strategies against CAA.

Investigating the ID3/SLC22A4 as immune-related signatures in ischemic stroke.

BACKGROUND: Ischemic stroke (IS) is a fearful disease that can cause a variety of immune events. Nevertheless, precise immune-related mechanisms have yet to be systematically elucidated. This study aimed to identify immune-related signatures using machine learning and to validate them with animal experiments and single cell analysis. METHODS: In this study, we screened 24 differentially expressed genes (DEGs) while identifying immune-related signatures that may play a key role in IS development through a comprehensive strategy between least absolute shrinkage and selection operation (LASSO) regression, support vector machine (SVM) and immune-related genes. In addition, we explored immune infiltration using the CIBERSORT algorithm. Finally, we performed validation in mouse brain tissue and single cell analysis. RESULTS: We identified 24 DEGs for follow-up analysis. ID3 and SLC22A4 were finally identified as the better immune-related signatures through a comprehensive strategy among DEGs, LASSO, SVM and immune-related genes. RT-qPCR, western blot, and immunofluorescence revealed a significant decrease in ID3 and a significant increase in SLC22A4 in the middle cerebral artery occlusion group. Single cell analysis revealed that ID3 was mainly concentrated in endothelial_2 cells and SLC22A4 in astrocytes in the MCAO group. A CIBERSORT finds significantly altered levels of immune infiltration in IS patients. CONCLUSIONS: This study focused on immune-related signatures after stroke and ID3 and SLC22A4 may be new therapeutic targets to promote functional recovery after stroke. Furthermore, the association of ID3 and SLC22A4 with immune cells may be a new direction for post-stroke immunotherapy.

ID3 regulates progesterone synthesis in bovine cumulus cells through modulation of mitochondrial function.

DNA binding inhibitory factor 3 (ID3) has been shown to have a key role in maintaining proliferation and differentiation. It has been suggested that ID3 may also affect mammalian ovarian function. However, the specific roles and mechanisms are unclear. In this study, the expression level of ID3 in cumulus cells (CCs) was inhibited by siRNA, and the downstream regulatory network of ID3 was uncovered by high-throughput sequencing. The effects of ID3 inhibition on mitochondrial function, progesterone synthesis, and oocyte maturation were further explored. The GO and KEGG analysis results showed that after ID3 inhibition, differentially expressed genes, including StAR, CYP11A1, and HSD3B1, were involved in cholesterol-related processes and progesterone-mediated oocyte maturation. Apoptosis in CC was increased, while the phosphorylation level of ERK1/2 was inhibited. During this process, mitochondrial dynamics and function were disrupted. In addition, the first polar body extrusion rate, ATP production and antioxidation capacity were reduced, which suggested that ID3 inhibition led to poor oocyte maturation and quality. The results will provide a new basis for understanding the biological roles of ID3 as well as cumulus cells.

A positive feedback loop between ID3 and PPARγ via DNA damage repair regulates the efficacy of radiotherapy for rectal cancer.

OBJECTIVE: To study the effect of inhibitor of differentiation 3 (ID3) on radiotherapy in patients with rectal cancer and to explore its primary mechanism. METHODS: Cell proliferation and clonogenic assays were used to study the relationship between ID3 and radiosensitivity. Co-immunoprecipitation and immunofluorescence were performed to analyze the possible mechanism of ID3 in the radiosensitivity of colorectal cancer. At the same time, a xenograft tumor model of HCT116 cells in nude mice was established to study the effect of irradiation on the tumorigenesis of ID3 knockdown colorectal cancer cells in vivo. Immunohistochemistry was performed to analyze the relationship between ID3 expression and the efficacy of radiotherapy in 46 patients with rectal cancer. RESULTS: Proliferation and clonogenic assays revealed that the radiosensitivity of colorectal cancer cells decreased with ID3 depletion through p53-independent pathway. With the decrease in ID3 expression, MDC1 was downregulated. Furthermore, the expression of ID3, MDC1, and γH2AX increased and formed foci after irradiation. ID3 interacted with PPARγ and form a positive feedback loop to enhance the effect of ID3 on the radiosensitivity of colorectal cancer. Irradiation tests in nude mice also confirmed that HCT116 cells with ID3 knockdown were more affected by irradiation. Immunohistochemical study showed that rectal cancer patients with low expression of ID3 had better radiotherapy efficacy. CONCLUSIONS: ID3 and PPARγ influence the radiosensitivity of colorectal cancer cells by interacting with MDC1 to form a positive feedback loop that promotes DNA damage repair. Patients with low expression of ID3 who received neoadjuvant chemoradiotherapy can obtain a better curative effect.

Análise imuno-histoquímica da Yes-Associated Protein (YAP) e sua influência sobre a proliferação celular e a apoptose em lesões odontogênicas epiteliais benignas / Immunohistochemical analysis of Yes-Associated Protein (YAP) and its influence on cell proliferation and apoptosis in benign epithelial odontogenic lesions

Introdução: Os cistos e tumores odontogênicos são lesões que apresentam comportamento biológico heterogêneo e patogênese ainda não totalmente esclarecida. A Yes-associated protein (YAP) atua como um regulador transcricional de genes envolvidos na proliferação celular e na apoptose, participando da ativação de vias associadas ao crescimento cístico e à progressão neoplásica. Objetivo: Analisar a expressão imuno-histoquímica da proteína YAP e correlacioná-la com marcadores envolvidos na proliferação celular e na apoptose em lesões odontogênicas epiteliais benignas. Metodologia: A amostra consistiu de 95 casos de lesões odontogênicas - 25 cistos dentígeros (CDs), 30 CO não sindrômicos (COs), 30 AMB convencionais (AMB-Cs) e 10 AMB unicísticos (AMB-Us) -, além de 10 espécimes de folículo dentários (FD). Foi realizada coleta dos dados clinico-demográficos dos casos, bem como análise morfológica para melhor caracterização da amostra. Os cortes histológicos foram submetidos à técnica imuno-histoquímica através da utilização dos anticorpos YAP, ciclina D1, Ki-67 e Bcl-2, e a análise da expressão destes foi realizada quali-quantitativamente, mediante metodologia adaptada. Os dados coletados seguiram para análise descritiva e estatística (p ≤ 0,05). Resultados: Houve discreta predileção por mulheres (n = 55; 57,6%) e por indivíduos na faixa etária dos 21 aos 40 anos (n = 50; 47,6%), sendo a região posterior de mandíbula mais afetada (64%). A análise da imunoexpressão de YAP revelou maiores níveis de expressão em COs, especialmente nas camadas basal e parabasal, seguido dos AMB-Us e AMB-Cs, que demonstraram moderada imunorreatividade, predominantemente nas células periféricas. Além disso, houve diferenças significativas quanto à imunoexpressão de YAP entre os grupos analisados, com existência de correlações positivas e estatisticamente significativas entre YAP e ciclina D1 em CDs e AMB-Us, e entre YAP e Ki-67 em AMB-Us (p < 0,05). Todavia, entre a imunoexpressão YAP e Bcl-2, foi verificada ausência de correlação estatisticamente significativa. Conclusões: A YAP pode exercer influência sobre a proliferação celular do epitélio de cistos e tumores odontogênicos, auxiliando, assim, na progressão das diferentes lesões odontogênicas (AU).

Background: Odontogenic cysts and tumors present heterogeneous biological behavior, and their etiopathogenesis is not fully understood yet. Yes-associated protein (YAP) acts as a transcriptional regulator of genes involved in cell proliferation and apoptosis, activating pathways associated with cystic growth and neoplastic progression. Objective: To analyze the immunohistochemical expression of YAP protein and correlate it with markers involved in cell proliferation and apoptosis in benign epithelial odontogenic lesions. Methods: The sample consisted of 95 cases of odontogenic lesions - 25 dentigerous cysts (DCs), 30 non-syndromic odontogenic keratocyst (OKCs), 30 conventional AMB (C-AMBs), and 10 unicystic AMB (UAMBs) -, in addition to 10 specimens of dental follicles (DF). Clinicodemographic data collection was carried out, as well as morphological analysis for better characterization of the sample. The histological sections were submitted to the immunohistochemical technique using YAP, cyclin D1, Ki-67, and Bcl-2 antibodies, and their immunoexpression analysis was performed qualitatively and quantitatively, through an adapted methodology. The collected data were submitted for descriptive and statistical analysis (p ≤ 0.05). Results: There was a slight predilection for women (n = 55; 57.6%) and individuals aged between 21 and 40 years (n = 50; 47.6%), with the posterior region of the mandible as the most affected site (64%). Analysis of YAP immunoexpression revealed higher expression levels in OKCs, especially in the basal and parabasal layers, followed by U-AMBs and C-AMBs, which showed moderate immunoreactivity, predominantly in peripheral cells. In addition, there were significant differences in YAP immunoexpression between the analyzed groups, with positive and statistically significant correlations between YAP and cyclin D1 in DCs and U-AMBs, and between YAP and Ki-67 in U-AMBs (p < 0.05). However, between YAP and Bcl-2 immunoexpression, there was no statistically significant correlation. Conclusions: YAP may influence on the cell proliferation of odontogenic cysts and tumors epithelium, thus helping with the progression of the different odontogenic lesions (AU) .

Inhibitor of DNA binding/differentiation proteins as IDs for pancreatic cancer: Role in pancreatic cancer initiation, development and prognosis.

A family of inhibitors of cell differentiation or DNA-binding proteins, known as ID proteins (ID1-4), function as mighty transcription factors in various cellular processes, such as inhibiting differentiation, promoting cell-cycle progression, senescence, angiogenesis, tumorigenesis, and metastasis in cancer. Pancreatic cancer represents the deadliest cancer with the lowest survival rate of 10% due to the diagnosis at an advanced fatal stage and therapeutic resistance. Modestly, the only curative option for this lethal cancer is surgery but is done in less than 15-20% of patients because of the locally aggressive and early metastatic nature. Finding the earliest biomarkers and targeting the various hallmarks of pancreatic cancer can improve the treatment and survival of pancreatic cancer patients. Therefore, herein in this review, we explore in depth the potential roles of ID proteins function in hallmarks of pancreatic cancer, signaling pathways, and its oncogenic and tumor-suppressive effects. Hence, understanding the roles of dysregulated ID proteins would provide new insights into its function in pancreatic cancer tumorigenesis.

ELK3-ID4 axis governs the metastatic features of triple negative breast cancer.

Purpose: Cancer cell metastasis is a multistep process, and the mechanism underlying extravasation remains unclear. ELK3 is a transcription factor that plays a crucial role in regulating various cellular processes, including cancer metastasis. Based on the finding that ELK3 promotes the metastasis of triple-negative breast cancer (TNBC), we investigated whether ELK3 regulates the extravasation of TNBC by forming the ELK3-ID4 axis. ID4 functions as a transcriptional regulator that interacts with other transcription factors, inhibiting their activity and subsequently influencing various biological processes associated with cell differentiation, survival, growth, and metastasis. Methods: We assessed the correlation between the expression of ELK3 and that of ID4 in TNBCs using bioinformatics analyses, QRT-PCR, western blot analysis, luciferase reporter assays, and chromatin immunoprecipitation. Migration, adhesion, invasion, and lung metastasis assays were employed to determine whether the ELK3-ID4 axis regulates the metastatic features of TNBC. Results: We found that ELK3 binds directly to a binding motif close to the ID4 promoter to repress promoter activity. The expression of E-cadherin in TNBC was regulated by the ELK3-ID4 axis. In vitro and in vivo analyses showed that inhibiting ID4 expression in ELK3-knockdown MDA-MB-231 (ELK3KD) cells restored the ability to extravasate and metastasize. Conclusion: The results indicate that the ELK3 regulates ID4 promoter activity, and that the ELK3-ID4 axis regulates the metastatic characteristics of TNBC cells. Additionally, the data suggest that the ELK3-ID4 axis regulates metastasis of TNBCs by modulating expression of E-cadherin.

Comprehensive analysis of ID genes reveals the clinical and prognostic value of ID3 expression in acute myeloid leukemia using bioinformatics identification and experimental validation.

BACKGROUND: Dysregulation of inhibitor of differentiation/DNA binding (ID) genes is linked to cancer growth, angiogenesis, invasiveness, metastasis and patient survival. Nevertheless, few investigations have systematically determined the expression and prognostic value of ID genes in acute myeloid leukemia (AML). METHODS: The expression and clinical prognostic value of ID genes in AML were first identified by public databases and further validated by our research cohort. RESULTS: Using public data, the expression of ID1/ID3 was markedly downregulated in AML, and the expression of ID2 was greatly upregulated in AML, whereas ID4 showed no significant difference. Among the ID genes, only ID3 expression may be the most valuable prognostic biomarker in both total AML and cytogenetically normal AML (CN-AML) and especially in CN-AML. Clinically, reduced ID3 expression was greatly associated with higher white blood cell counts, peripheral blood/bone marrow blasts, normal karyotypes and intermediate cytogenetic risk. In addition, low ID3 expression was markedly related to FLT3 and NPM1 mutations as well as wild-type TP53. Despite these associations, multivariate Cox regression analysis revealed that ID3 expression was an independent risk factor affecting overall survival (OS) and disease free survival (DFS) in CN-AML patients. Biologically, a total of 839 mRNAs/lncRNAs and 72 microRNAs were found to be associated with ID3 expression in AML. Importantly, the expression of ID3 with discriminative value in AML was further confirmed in our research cohort. CONCLUSION: The bioinformatics analysis and experimental verification demonstrate that low ID3 expression independently affects OS and DFS in patients with CN-AML, which might be seen as a potential prognostic indicator in CN-AML.

miR-210-3p protects against osteoarthritis through inhibiting subchondral angiogenesis by targeting the expression of TGFBR1 and ID4.

Excessive subchondral angiogenesis is a key pathological feature of osteoarthritis (OA), as it alters the balance of subchondral bone remodeling and causes progressive cartilage degradation. We previously found that miR-210-3p correlates negatively with angiogenesis, though the specific mechanism of miR-210-3p-related angiogenesis in subchondral bone during OA progression remains unclear. This study was conducted to identify the miR-210-3p-modulating subchondral angiogenesis mechanism in OA and investigate its therapeutic effect. We found that miR-210-3p expression correlated negatively with subchondral endomucin positive (Emcn+) vasculature in the knee joints of OA mice. miR-210-3p overexpression regulated the angiogenic ability of endothelial cells (ECs) under hypoxic conditions in vitro. Mechanistically, miR-210-3p inhibited ECs angiogenesis by suppressing transforming growth factor beta receptor 1 (TGFBR1) mRNA translation and degrading DNA-binding inhibitor 4 (ID4) mRNA. In addition, TGFBR1 downregulated the expression of ID4. Reduced ID4 levels led to a negative feedback regulation of TGFBR1, enhancing the inhibitory effect of miR-210-3p on angiogenesis. In OA mice, miR-210-3p overexpression in ECs via adeno-associated virus (AAV) alleviated cartilage degradation, suppressed the type 17 immune response and relieved symptoms by attenuating subchondral Emcn+ vasculature and subchondral bone remodeling. In conclusion, we identified a miR-210-3p/TGFBR1/ID4 axis in subchondral ECs that modulates OA progression via subchondral angiogenesis, representing a potential OA therapy target.

Shifting gears: Id3 enables recruitment of E proteins to new targets during T cell development and differentiation.

Shifting levels of E proteins and Id factors are pivotal in T cell commitment and differentiation, both in the thymus and in the periphery. Id2 and Id3 are two different factors that prevent E proteins from binding to their target gene cis-regulatory sequences and inducing gene expression. Although they use the same mechanism to suppress E protein activity, Id2 and Id3 play very different roles in T cell development and CD4 T cell differentiation. Id2 imposes an irreversible choice in early T cell precursors between innate and adaptive lineages, which can be thought of as a railway switch that directs T cells down one path or another. By contrast, Id3 acts in a transient fashion downstream of extracellular signals such as T cell receptor (TCR) signaling. TCR-dependent Id3 upregulation results in the dislodging of E proteins from their target sites while chromatin remodeling occurs. After the cessation of Id3 expression, E proteins can reassemble in the context of a new genomic landscape and molecular context that allows induction of different E protein target genes. To describe this mode of action, we have developed the "Clutch" model of differentiation. In this model, Id3 upregulation in response to TCR signaling acts as a clutch that stops E protein activity ("clutch in") long enough to allow shifting of the genomic landscape into a different "gear", resulting in accessibility to different E protein target genes once Id3 decreases ("clutch out") and E proteins can form new complexes on the DNA. While TCR signal strength and cytokine signaling play a role in both peripheral and thymic lineage decisions, the remodeling of chromatin and E protein target genes appears to be more heavily influenced by the cytokine milieu in the periphery, whereas the outcome of Id3 activity during T cell development in the thymus appears to depend more on the TCR signal strength. Thus, while the Clutch model applies to both CD4 T cell differentiation and T cell developmental transitions within the thymus, changes in chromatin accessibility are modulated by biased inputs in these different environments. New emerging technologies should enable a better understanding of the molecular events that happen during these transitions, and how they fit into the gene regulatory networks that drive T cell development and differentiation.

The divergence between T cell and innate lymphoid cell fates controlled by E and Id proteins.

T cells develop in the thymus from lymphoid primed multipotent progenitors or common lymphoid progenitors into αß and γδ subsets. The basic helix-loop-helix transcription factors, E proteins, play pivotal roles at multiple stages from T cell commitment to maturation. Inhibitors of E proteins, Id2 and Id3, also regulate T cell development while promoting ILC differentiation. Recent findings suggest that the thymus can also produce innate lymphoid cells (ILCs). In this review, we present current findings that suggest the balance between E and Id proteins is likely to be critical for controlling the bifurcation of T cell and ILC fates at early stages of T cell development.

Id3 expression identifies CD4+ memory Th1 cells.

Memory CD4+ T cells play a pivotal role in mediating long-term protective immunity, positioning them as an important target in vaccine development. However, multiple functionally distinct helper CD4+ T-cell subsets can arise in response to a single invading pathogen, complicating the identification of rare populations of memory precursor cells during the effector phase of infection and memory CD4+ T cells following pathogen clearance and the contraction phase of infection. Furthermore, current literature remains unclear regarding whether a single CD4+ memory T-cell lineage gives rise to secondary CD4+ T helper subsets or if there are unique memory precursor cells within each helper lineage. A majority of T follicular helper (Tfh) cells, which have established memory potential, express Id3, an inhibitor of E protein transcription factors, following acute viral infection. We show that expression of Id3 definitively identified a subset of cells within both the CD4+ Tfh and T helper 1 (Th1) lineages at memory time points that exhibited memory potential, with the capacity for significant re-expansion in response to secondary infection. Notably, we demonstrate that a subset of Th1 cells that survive into the memory phase were marked by Id3 expression and possessed the potential for enhanced expansion and generation of both Th1 and Tfh secondary effector cell populations in a secondary response to pathogen. Additionally, these cells exhibited enrichment of key molecules associated with memory potential when compared with Id3lo Th1 cells. Therefore, we propose that Id3 expression serves as an important marker to indicate multipotent potential in memory CD4+ T cells.