Inhibitor Of Differentiation-3 Research Articles

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

ObjectiveTo study the effect of inhibitor of differentiation 3 (ID3) on radiotherapy in patients with rectal cancer and to explore its primary mechanism.MethodsCell 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.ResultsProliferation 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.ConclusionsID3 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.

MicroRNA-24-3p promotes skeletal muscle differentiation and regeneration by regulating HMGA1.

Numerous studies have established the critical roles of microRNAs in regulating post-transcriptional gene expression in diverse biological processes. Here, we report on the role and mechanism of miR-24-3p in skeletal muscle differentiation and regeneration. miR-24-3p promotes myoblast differentiation and skeletal muscle regeneration by directly targeting high mobility group AT-hook 1 (HMGA1) and regulating it and its direct downstream target, the inhibitor of differentiation 3 (ID3). miR-24-3p knockdown in neonatal mice increases PAX7-positive proliferating muscle stem cells (MuSCs) by derepressing Hmga1 and Id3. Similarly, inhibition of miR-24-3p in the tibialis anterior muscle prevents Hmga1 and Id3 downregulation and impairs regeneration. These findings provide evidence that the miR-24-3p/HMGA1/ID3 axis is required for MuSC differentiation and skeletal muscle regeneration in vivo.

B-1b Cells Possess Unique bHLH-Driven P62-Dependent Self-Renewal and Atheroprotection.

B1a and B1b lymphocytes produce IgM that inactivates oxidation-specific epitopes (IgMOSE) on LDL (low-density lipoprotein) and protects against atherosclerosis. Loss of ID3 (inhibitor of differentiation 3) in B cells selectively promotes B1b but not B1a cell numbers, leading to higher IgMOSE production and reduction in atherosclerotic plaque formation. Yet, the mechanism underlying this regulation remains unexplored. Bulk RNA sequencing was utilized to identify differentially expressed genes in B1a and B1b cells from Id3KO and Id3WT mice. CRISPR/Cas9 and lentiviral genome editing coupled with adoptive transfer were used to identify key Id3-dependent signaling pathways regulating B1b cell proliferation and the impact on atherosclerosis. Biospecimens from humans with advanced coronary artery disease imaging were analyzed to translate murine findings to human subjects with coronary artery disease. Through RNA sequencing, P62 was found to be enriched in Id3KO B1b cells. Further in vitro characterization reveals a novel role for P62 in mediating BAFF (B-cell activating factor)-induced B1b cell proliferation through interacting with TRAF6 (tumor necrosis factor receptor 6) and activating NF-κB (nuclear factor kappa B), leading to subsequent C-MYC (C-myelocytomatosis) upregulation. Promoter-reporter assays reveal that Id3 inhibits the E2A protein from activating the P62 promoter. Mice adoptively transferred with B1 cells overexpressing P62 exhibited an increase in B1b cell number and IgMOSE levels and were protected against atherosclerosis. Consistent with murine mechanistic findings, P62 expression in human B1 cells was significantly higher in subjects harboring a function-impairing single nucleotide polymorphism (SNP) at rs11574 position in the ID3 gene and directly correlated with plasma IgMOSE levels. This study unveils a novel role for P62 in driving BAFF-induced B1b cell proliferation and IgMOSE production to attenuate diet-induced atherosclerosis. Results identify a direct role for Id3 in antagonizing E2A from activating the p62 promoter. Moreover, analysis of putative human B1 cells also implicates these pathways in coronary artery disease subjects, suggesting P62 as a new immunomodulatory target for treating atherosclerosis.

ID3 mediates the TGF-β1-induced suppression of matrix metalloproteinase-1 in human granulosa cells.

In the mammalian ovary, matrix metalloproteinase-1 (MMP1) is expressed in growing ovarian follicles, and MMP1-mediated extracellular matrix (ECM) remodeling plays a functional role in regulating the formation of corpus luteum. Transforming growth factor-β1 (TGF-β1) is an intraovarian growth factor that acts as a negative regulator of luteinization and progesterone production in human granulosa-lutein (hGL) cells. At present, whether TGF-β1 regulates the expression of MMP1 and thus affects ECM remodeling during corpus luteum formation remains largely unknown. The aim of this study was to investigate the effects of TGF-β1 and the molecular mechanisms by which it regulates the expression of MMP1 in immortalized human granulosa cells lines (SVOG) and primary hGL cells (obtained from consenting patients undergoing IVF treatment). We used inhibition approaches including a competitive antagonist for endogenous TGF-β type II receptor, pharmacological inhibitors (SB431542 and dorsomorphin), and specific small interfering RNA-targeted knockdown of ALK5 type I receptor and SMAD4 to demonstrate that TGF-β1 downregulates the expression and production of MMP1 via a TβRII/ALK5-mediated SMAD-dependent signaling pathway in hGL cells. Additionally, our results show that the suppressive effect of TGF-β1 on the expression of MMP1 is mediated by a transcription factor, the inhibitor of differentiation 3 (ID3) protein. Our findings provide insights into the molecular interactions and mechanisms of TGF-β1 and ID3 during the regulation of MMP1 in hGL cells.

ID3 Promotes Stem Cell Features and Predicts Chemotherapeutic Response of Intrahepatic Cholangiocarcinoma.

Cancer stem cells contribute to a high rate of recurrence and chemotherapeutic resistance in many types of cancer, including intrahepatic cholangiocarcinoma (ICC). Inhibitor of differentiation 3 (ID3) has been reported to promote cancer stem cells, but its role in ICC is obscure. In this study, we identified that ID3 is highly expressed in human ICC tissues compared with matched normal tissues and correlates with poor prognosis. Functional studies demonstrate that ID3 is required for stemness maintenance in cholangiocarcinoma both in vitro and in vivo. Consistent with the regulation of cancer stem cell features by ID3, transgenic expression of ID3 enhances chemoresistance of cholangiocarcinoma cells. Moreover, we found that ICC patients with low ID3 levels benefited from postoperative transarterial chemoembolization, whereas patients with high ID3 levels did not, indicating the significance of ID3 in individualized ICC therapy. Mechanistically, ID3 could interact with E47 and block E47 recruitment to the promoter of β-catenin, which leads to activation of Wnt/β-catenin signaling. Conclusion: Our results show that ID3 could promote the stemness of ICC by increasing the transcriptional activity of β-catenin and could serve as a biomarker in predicting ICC patients' response to adjuvant chemotherapeutics.

Abstract 614: From Genotype to Phenotype: Molecular and Functional Characterization of the CVD-associated SNP rs11574 in ID3

Atherosclerosis is the primary pathological process of CVD and is strongly controlled by heritable factors; however, the mechanisms and pathways through which many of these genetic components regulate plaque development are poorly understood. Recently, a SNP in the coding region of the gene inhibitor of differentiation 3 ( ID3 ) at rs11574 was identified in multiple studies to be associated with CVD. Mutation of rs11574 from the major allele (G) to the minor allele (A) changes the 105 th amino acid of ID3 from an alanine to a threonine and is associated with an attenuated ability of ID3 to antagonize bHLH transcription factors and to prevent them from binding to DNA and activating transcription. The current study utilized co-immunoprecipitation to demonstrate that the minor allele of rs11574 specifically impairs the ability of ID3 to bind to and sequester E12 and no other member of the bHLH family. ChIP studies further demonstrated that the impaired regulatory ability of the ID3 minor allele variant increases E12 occupancy at promoter regions and enhances transcription of E12 target genes including smooth muscle alpha actin ( Acta2 ) and p21 in murine vascular smooth muscle cells (VSMCs). To study the role of this SNP in human cells in intact chromatin under endogenous regulation, we genome edited human 293T cells using CRISPR/Cas9 to produce the allelic variants of rs11574 in the ID3 gene (GG, AG, and AA). Edited cells showed significant changes in cellular proliferation, gene expression, and promoter occupancy depending upon genotype at rs11574. Cells containing the minor allele of rs11574 displayed significantly increased p21 expression and proliferated more slowly than cells containing the major allele of rs11574; whereas, cells heterozygote at rs11574 displayed an intermediate phenotype. These data implicate rs11574 in the regulation of cellular proliferation and mature VSMC marker expression, key phenotypes regulated in the processes of lesion development and formation.

Prospective Studies Exploring the Possible Impact of an ID3 Polymorphism on Changes in Obesity Measures.

Changes in fat mass depend on adipogenesis and angiogenesis, mechanisms regulated by the inhibitor of differentiation-3 (ID3). Id3 knockout mice showed attenuated increases in BMI and visceral fat mass. We hypothesized that the ID3 missense variant (rs11574-A) would lead to an attenuated increase over time in fat mass, BMI, waist circumference (WC), and waist-hip ratio (WHR) in humans. The genotyped study populations included the Obesity Research Group - Genetics (ORGGEN) cohort, a cohort of men with obesity (N = 716) and of randomly selected men (N = 826) from the Danish draft register who were examined at mean ages of 20 and 46 years, and the Inter99 (N = 6,116) and Health2006 (N = 2,761) cohorts, two population-based samples of middle-aged people, followed up after 5 years. In meta-analyses of all data, no association was found between rs11574-A and changes in BMI, WC, WHR, or fat mass. We found an association between rs11574-A and cross-sectional BMI (N = 10,359, β: -0.16 kg/m2 per allele, 95% CI: -0.30 to -0.01, P = 0.033) and fat mass (N = 4,188, β: -0.52 kg/m2 per allele, 95% CI: -1.03 to -0.01, P = 0.046). No consistent impact of the genetic variant on changes in fat mass, BMI, or fat distribution was found in three Danish cohorts.

Inhibitor of Differentiation-3 and Estrogenic Endocrine Disruptors: Implications for Susceptibility to Obesity and Metabolic Disorders.

The rising global incidence of obesity cannot be fully explained within the context of traditional risk factors such as an unhealthy diet, physical inactivity, aging, or genetics. Adipose tissue is an endocrine as well as a metabolic organ that may be susceptible to disruption by environmental estrogenic chemicals. Since some of the endocrine disruptors are lipophilic chemicals with long half-lives, they tend to bioaccumulate in the adipose tissue of exposed populations. Elevated exposure to these chemicals may predispose susceptible individuals to weight gain by increasing the number and size of fat cells. Genetic studies have demonstrated that the transcriptional regulator inhibitor of differentiation-3 (ID3) promotes high fat diet-induced obesity in vivo. We have shown previously that PCB153 and natural estrogen 17β-estradiol increase ID3 expression. Based on our findings, we postulate that ID3 is a molecular target of estrogenic endocrine disruptors (EEDs) in the adipose tissue and a better understanding of this relationship may help to explain how EEDs can lead to the transcriptional programming of deviant fat cells. This review will discuss the current understanding of ID3 in excess fat accumulation and the potential for EEDs to influence susceptibility to obesity or metabolic disorders via ID3 signaling.

Targeting Angiogenesis as Treatment for Obesity

Worldwide, more than 1 billion adults are overweight, and obesity constitutes a key contributor to chronic disease. The conventional wisdom is that this rising epidemic reflects societal adaptation to a sedentary lifestyle and nutritional transition to overconsumption of inexpensive and calorie-dense diets. Implementing behavioral lifestyle modifications to promote weight loss and maintenance, including physical activity and a healthy diet, constitutes a major clinical challenge and remains difficult to achieve. Similarly, there is concern about the safety and potential long-term complications associated with enforcing caloric restriction through bariatric surgery. Finally, the currently available two pharmacological agents, orlistat and phentermine, have limited efficacy and side effects. Consequently, there is an unprecedented need for the development of effective and safe weight loss strategies, and understanding the physiology of obesity may provide novel therapeutic opportunities. See accompanying article on page 317 In the current issue, Cutchins et al identify a previously unrecognized mechanism underlying adipose tissue expansion during obesity.1 The authors first demonstrate that the inhibitor of differentiation-3 (Id3) is expressed in adipose tissue and induced during obesity. Fractionation and immunohistochemistry of the adipose tissue revealed that high-fat diet feeding and the ensuing development of obesity results in increased Id3 expression in endothelial cells. Important functional experiments demonstrated that genetic deletion of Id3 in mice prevents adipose tissue expansion during …

EGFR-AKT-Smad Signaling Promotes Formation of Glioma Stem-like Cells and Tumor Angiogenesis by ID3-Driven Cytokine Induction

Aberrant activation of receptor tyrosine kinases (RTK) is causally linked to the pathobiological traits of glioblastoma and genesis of glioma stem-like cells (GSC), but the underlying mechanism is still unknown. Here, we show that epidermal growth factor receptor (EGFR) signaling regulates the proliferation, angiogenesis, and acquisition of GSC characteristics by inducing inhibitor of differentiation 3 (ID3) and ID3-regulated cytokines [GRO1 and interleukins (IL)-6 and 8] induction. We found that EGFR-mediated ID3 expression was regulated by Smad5, which was directly phosphorylated by AKT. Furthermore, ID3 alone imparted GSC features to primary astrocytes derived from Ink4a/Arf-deficient mouse, and EGFR-ID3-IL-6 signaling axis gave rise to tumor cell heterogeneity. Conversely, EGFR inhibitors suppressed EGFR-AKT-Smad5-driven induction of ID3, which led to a decrease in the tumorsphere forming ability of GSCs and U87MG cells that possess an active mutant EGFR, EGFRvIII, without obvious cytotoxic effects. However, these cells seemed to regain colonogenic ability after removal of the EGFR inhibitors. Together, the results delineate a novel integrative molecular mechanism in which the RTK-ID signaling pathway governs genesis and maintenance of GBM histopathologic features, such as GSCs-based tumor initiation, progression, and angiogenesis.

B-Cell Aortic Homing and Atheroprotection Depend on Id3

B cells are abundant in the adventitia of normal and diseased vessels. Yet, the molecular and cellular mechanisms mediating homing of B cells to the vessel wall and B-cell effects on atherosclerosis are poorly understood. Inhibitor of differentiation-3 (Id3) is important for atheroprotection in mice and polymorphism in the human ID3 gene has been implicated as a potential risk marker of atherosclerosis in humans. Yet, the role of Id3 in B-cell regulation of atherosclerosis is unknown. To determine if Id3 regulates B-cell homing to the aorta and atheroprotection and identify molecular and cellular mechanisms mediating this effect. Loss of Id3 in Apoe(-/-) mice resulted in early and increased atherosclerosis. Flow cytometry revealed a defect in Id3(-/-) Apoe(-/-) mice in the number of B cells in the aorta but not the spleen, lymph nodes, and circulation. Similarly, B cells transferred from Id3(-/-) Apoe(-/-) mice into B-cell-deficient mice reconstituted spleen, lymph node, and blood similarly to B cells from Id3(+/+) Apoe(-/-) mice, but aortic reconstitution and B-cell-mediated inhibition of diet-induced atherosclerosis was significantly impaired. In addition to retarding initiation of atherosclerosis, B cells homed to regions of existing atherosclerosis, reduced macrophage content in plaque, and attenuated progression of disease. The chemokine receptor CCR6 was identified as an important Id3 target mediating aortic homing and atheroprotection. Together, these results are the first to identify the Id3-CCR6 pathway in B cells and demonstrate its role in aortic B-cell homing and B-cell-mediated protection from early atherosclerosis.

Id3 Is a Novel Atheroprotective Factor Containing a Functionally Significant Single-Nucleotide Polymorphism Associated With Intima–Media Thickness in Humans

The gene encoding the helix-loop-helix transcription factor Id3 (inhibitor of differentiation-3) is located within atherosclerosis susceptibility loci of both mice and humans, yet its influence on atherosclerosis is not known. The present study sought to determine whether polymorphisms in the ID3 gene were associated with indices of atherosclerosis in humans and if loss of Id3 function modulated atherogenesis in mice. Six tagging single-nucleotide polymorphisms (SNPs) (tagSNPs) in the human ID3 gene were assessed in participants of the Diabetes Heart Study. One tagSNP, rs11574, was independently associated with carotid intima-media thickness (IMT). The human ID3 variant at rs11574 results in an alanine to threonine substitution in the C terminus. To determine the effect of this polymorphism on the basic function of Id3, site-directed mutagenesis of the human ID3 gene at rs11574 was performed. Results demonstrated a significant reduction in coimmunoprecipitation of the known E-protein partner, E12, with Id3 when it contains the sequence encoded by the risk allele (Id3105T). Further, Id3105T had an attenuated ability to modulate E12-mediated transcriptional activation compared to Id3 containing the ancestral allele (Id3105A). Microarray analysis of vascular smooth muscle cells from WT and Id3(-/-) mice revealed significant modulation of multiple gene pathways implicated in atherogenesis. Moreover, Id3(-/-)ApoE(-/-) mice developed significantly more atherosclerosis in response to 32 weeks of Chow or Western diet feeding than Id3(+/+)ApoE(-/-) mice. Taken together, results provide novel evidence that Id3 is an atheroprotective factor and link a common SNP in the human ID3 gene to loss of Id3 function and increased IMT.

A novel EID family member, EID-3, inhibits differentiation and forms a homodimer or heterodimer with EID-2

The EID family members, i.e., E1A-like inhibitor of differentiation-1 (EID-1) and EID-1-like inhibitor of differentiation-2 (EID-2), were identified as negative regulators of cellular differentiation. EID-1 seems to inhibit differentiation by blocking histone acetyltransferase activity and EID-2 possibly inhibits differentiation through binding to class I histone deacetylases (HDACs). Here, we report a novel inhibitor of differentiation exhibiting homology with EID-2 termed EID-3 (EID-2-like inhibitor of differentiation-3). Like EID-2, EID-3 inhibited MyoD- and GRα-dependent transcription and blocked muscle differentiation in cultured cells by binding to class I HDACs. Unlike that of EID-2, the C-terminus, but not the N-terminus, of EID-3 was required for nuclear localization. EID-3 formed a homodimer or heterodimer with EID-2. These results suggest that EID-3 inhibits differentiation by blocking transcription as a complex in cells.