Inhibitor Of DNA Binding Proteins Research Articles

Amphiregulin stimulates human chorionic gonadotropin expression by inducing ERK1/2-mediated ID3 expression in trophoblast cells

IntroductionThe human chorionic gonadotropin (hCG) is a dimer consisting of an α subunit and a β subunit which is encoded by the CGB gene and is unique to hCG. hCG is a hormone mainly synthesized by syncytiotrophoblast cells in the placenta, plays a critical role in stimulating progesterone production that is necessary for maintaining normal pregnancy in the early stage. Epidermal growth factor receptor (EGFR) belongs to the receptor tyrosine kinase family which has been shown to regulate various physiological and pathological events. In human chorionic villi and amniotic fluid, amphiregulin (AREG) is reported to be the most abundant EGFR ligand and can stimulate hCG expression. However, the underlying mechanism remains unknown. MethodsWe use BeWo cells, the commonly used cell model for the hCG production of trophoblast cells, as an in vitro model. The effects of AREG on CGB expression and hCG secretion as well as the underlying mechanisms were explored by a series of in vitro experiments. ResultsWe show that treatment with AREG stimulates CGB expression and hCG secretion. Using pharmacological inhibitors, we show that the stimulatory effects of AREG on CGB expression and hCG secretion are mediated by the EGFR-activated ERK1/2 signaling pathways. In addition, the expression of inhibitor of DNA-binding protein 3 (ID3) is upregulated by AREG. Knockdown of ID3 attenuates the AREG-induced upregulation of CGB expression and hCG secretion. DiscussionThis study provides important insights into the molecular mechanisms that mediate AREG-induced upregulation of hCG production in human trophoblast cells which may lead to the development of alternative therapeutic approaches for the treatment of placental diseases.

Id1 Ablation Protects Hematopoietic Stem Cells from Stress-Induced Exhaustion and Aging

Defining mechanisms that maintain tissue stem cells during homeostasis and stress is important for improving tissue regeneration and repair, cancer and aging. Inhibitor of DNA binding protein (Id1) is induced in hematopoietic stem cells (HSCs) by proinflammatory cytokines that promote HSC proliferation and differentiation in vitro and in vivo, suggesting that Id1 may function during stress hematopoiesis. Id1-/- HSCs show increased self-renewal in serial bone marrow transplantation (BMT) assays, which is correlated with decreased proliferation, mitochondrial biogenesis, and ROS production. As a result, Id1-/- HSCs harbor less DNA damage, show increased quiescence and reduced aging. Mechanistically, we show that cytokines and other proinflammatory stimuli present in the hematopoietic microenvironment after γ-irradiation induce Id1 expression. Id1-/- HSCs fail to properly respond to cytokine-induced proliferation, suggesting that HSC exhaustion is mediated, in part, by chronic proliferative stress. Id1-/- HSCs are protected from other chronic proliferative stresses including chronic genotoxic and inflammatory stress, and aging. Thus, targeting Id1 inhibition may be therapeutically useful to improve HSC survival and function during BMT, chronic stress and aging. Funded by FNLCR Contract HHSN261200800001E DisclosuresNo relevant conflicts of interest to declare.

EPCT-07. ID1 IS A KEY TRANSCRIPTIONAL REGULATOR OF DIPG INVASION AND IS TARGETABLE WITH CANNABIDIOL

Diffuse intrinsic pontine gliomas (DIPGs) are lethal pediatric brain tumors with no effective therapies beyond radiation. The highly invasive nature of DIPG is key to its aggressive phenotype, but the factors and mechanisms contributing to this aggressive invasion are unknown. Inhibitor of DNA binding (ID) proteins, key regulators of lineage commitment during embryogenesis, are implicated in tumorigenesis in multiple human solid tumors. Prior work showed that recurrent H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes. However, the impact and targetability of ID1 have not been explored in human DIPG. Exome and transcriptome sequencing analyses of multi-focal DIPG tumors and normal brain tissue from autopsy (n=52) revealed that ID1 expression is significantly elevated in DIPG samples. Higher ID1 expression correlates with reduced survival in DIPG patients and increased regional invasion in multi-focal autopsy samples. Analyses of developing mouse brain RNA/ChIP-Seq data revealed high ID1 expression and H3K27ac promoter binding in prenatal hindbrain compared to all other prenatal and postnatal brain regions. ChIP-qPCR for H3K27ac and H3K27me3 revealed that ID1 gene regulatory regions are epigenetically poised for upregulation in DIPG tissues compared to normal brain, regardless of H3/ACVR1 mutational status. These data support that the developing pons is regionally poised for ID1 activation. Genetic (shRNA) ID1 knockdown of primary human H3.3K27M-DIPG cells (DIPG007) resulted in significantly reduced invasion/migration and significantly improved survival of K27M-DIPG mice. Knockdown of ID1 in DIPG cells also resulted in down-regulation of the WNK1-NKCC1 pathway, which regulates tumor cell electrolyte homeostasis and migration. Finally, treatment of DIPG007 cells with cannabidiol (CBD) reduced ID1 levels, viability of DIPG cells and significantly improved survival of K27M-DIPG mice. In summary, our findings indicate that multifactorial (genetic and regional) epigenetic upregulation of ID1 drives DIPG invasiveness; and that targeting ID1 with CBD could potentially be an effective therapy for DIPG.

ID1 and ID3 are Negative Regulators of TGFβ2-Induced Ocular Hypertension and Compromised Aqueous Humor Outflow Facility in Mice.

PurposeIn POAG, elevated IOP remains the major risk factor in irreversible vision loss. Increased TGFβ2 expression in POAG aqueous humor and in the trabecular meshwork (TM) amplifies extracellular matrix (ECM) deposition and reduces ECM turnover in the TM, leading to a decreased aqueous humor (AH) outflow facility and increased IOP. Inhibitor of DNA binding proteins (ID1 and ID3) inhibit TGFβ2-induced fibronectin and PAI-1 production in TM cells. We examined the effects of ID1 and ID3 gene expression on TGFβ2-induced ocular hypertension and decreased AH outflow facility in living mouse eyes.MethodsIOP and AH outflow facility changes were determined using a mouse model of Ad5-hTGFβ2C226S/C288S-induced ocular hypertension. The physiological function of ID1 and ID3 genes were evaluated using Ad5 viral vectors to enhance or knockdown ID1/ID3 gene expression in the TM of BALB/cJ mice. IOP was measured in conscious mice using a Tonolab impact tonometer. AH outflow facilities were determined by constant flow infusion in live mice.ResultsOver-expressing ID1 and ID3 significantly blocked TGFβ2-induced ocular hypertension (P < 0.0001). Although AH outflow facility was significantly decreased in TGFβ2-transduced eyes (P < 0.04), normal outflow facility was preserved in eyes injected concurrently with ID1 or ID3 along with TGFβ2. Knockdown of ID1 or ID3 expression exacerbated TGFβ2-induced ocular hypertension.ConclusionsIncreased expression of ID1 and ID3 suppressed both TGFβ2-elevated IOP and decreased AH outflow facility. ID1 and/or ID3 proteins thus may show promise as future candidates as IOP-lowering targets in POAG.

BMP9-ID1 signaling promotes EpCAM-positive cancer stem cell properties in hepatocellular carcinoma.

The malignant nature of hepatocellular carcinoma (HCC) is closely related to the presence of cancer stem cells (CSCs). Bone morphologic protein 9 (BMP9), a member of the transforming growth factor‐beta (TGF‐β) superfamily, was recently reported to be involved in liver diseases including cancer. We aimed to elucidate the role of BMP9 signaling in HCC‐CSC properties and to assess the therapeutic effect of BMP receptor inhibitors in HCC. We have identified that high BMP9 expression in tumor tissues or serum from patients with HCC leads to poorer outcome. BMP9 promoted CSC properties in epithelial cell adhesion molecule (EpCAM)‐positive HCC subtype via enhancing inhibitor of DNA‐binding protein 1 (ID1) expression in vitro. Additionally, ID1 knockdown significantly repressed BMP9‐promoted HCC‐CSC properties by suppressing Wnt/β‐catenin signaling. Interestingly, cells treated with BMP receptor inhibitors K02288 and LDN‐212854 blocked HCC‐CSC activation by inhibiting BMP9‐ID1 signaling, in contrast to cells treated with the TGF‐β receptor inhibitor galunisertib. Treatment with LDN‐212854 suppressed HCC tumor growth by repressing ID1 and EpCAM in vivo. Our study demonstrates the pivotal role of BMP9‐ID1 signaling in promoting HCC‐CSC properties and the therapeutic potential of BMP receptor inhibitors in treating EpCAM‐positive HCC. Therefore, targeting BMP9‐ID1 signaling could offer novel therapeutic options for patients with malignant HCC.

Id4 Suppresses the Growth and Invasion of Colorectal Cancer HCT116 Cells through CK18-Related Inhibition of AKT and EMT Signaling.

Id4 is one of the inhibitors of DNA-binding proteins (Id) and involved in the pathogenesis of numerous cancers. The specific mechanism underlying the Id4-mediated regulation of proliferation, invasion, and metastasis of colorectal cancer (CRC) cells is still largely unclear. In the present study, results showed CRC cells had a lower baseline Id4 expression than normal intestinal epithelial NCM460 cells. In order to explore the role of Id4 in the tumorigenicity, CRC HCT116 cells with stable Id4 expression were used, and results showed Id4 overexpression arrested the cell cycle at the G0/G1 phase, inhibited the cell proliferation and the colony formation, as well as suppressed the migration and invasion. In the in vivo model, Id4 overexpression inhibited the tumor growth and metastasis in the nude mice. Furthermore, Id4 overexpression upregulated the expression of proteins associated with cell proliferation, inhibited the PI3K/AKT pathway, and suppressed epithelial-mesenchymal transition (EMT) of HCT116 cells. Moreover, Id4 significantly decreased cytokeratin 18 (CK18) expression, but CK18 overexpression in Id4 expressing HCT116-Id4 cells rescued the activation of AKT, p-AKT, MMP2, MMP7, and E-cadherin. Collectively, our study indicated Id4 may inhibit CRC growth and metastasis through inhibiting the PI3K/AKT pathway in a CK18-dependent manner and suppressing EMT. Id4 may become a target for the treatment of CRC.

TGF-β-Induced Endothelial to Mesenchymal Transition Is Determined by a Balance Between SNAIL and ID Factors.

Endothelial-to-mesenchymal transition (EndMT) plays an important role in embryonic development and disease progression. Yet, how different members of the transforming growth factor-β (TGF-β) family regulate EndMT is not well understood. In the current study, we report that TGF-β2, but not bone morphogenetic protein (BMP)9, triggers EndMT in murine endothelial MS-1 and 2H11 cells. TGF-β2 strongly upregulates the transcription factor SNAIL, and the depletion of Snail is sufficient to abrogate TGF-β2-triggered mesenchymal-like cell morphology acquisition and EndMT-related molecular changes. Although SLUG is not regulated by TGF-β2, knocking out Slug also partly inhibits TGF-β2-induced EndMT in 2H11 cells. Interestingly, in addition to SNAIL and SLUG, BMP9 stimulates inhibitor of DNA binding (ID) proteins. The suppression of Id1, Id2, or Id3 expression facilitated BMP9 in inducing EndMT and, in contrast, ectopic expression of ID1, ID2, or ID3 abrogated TGF-β2-mediated EndMT. Altogether, our results show that SNAIL is critical and indispensable for TGF-β2-mediated EndMT. Although SLUG is also involved in the EndMT process, it plays less of a crucial role in it. In contrast, ID proteins are essential for maintaining endothelial traits and repressing the function of SNAIL and SLUG during the EndMT process. These data suggest that the control over endothelial vs. mesenchymal cell states is determined, at least in part, by a balance between the expression of SNAIL/SLUG and ID proteins.

Micellar Casein and Whey Powder Hold a TGF-β Activity and Regulate ID Genes In Vitro.

Casein and whey being food supplements have been considered to be used in oral health care products. However, the response of oral cells to micellar casein and whey powder remains unclear. Considering that milk contains the growth factor TGF-β, and lactoperoxidase was recently reported to decrease the expression of inhibitor of DNA-binding (ID) proteins, there is a rationale to assume that casein and whey can also provoke these responses in oral cells. To examine the TGF-β activity, gingival fibroblasts were exposed to reconstituted casein and whey powder from food supplement before the expression of TGF-β target genes were analyzed by reverse transcription-quantitative polymerase chain reaction. Immunoassays were performed for interleukin11 (IL11) in the cell culture supernatant and for TGF-β in the reconstituted casein and whey. We blocked TGF-β by neutralizing the antibody and the TGF-β receptor type I kinase with the inhibitor SB431542. We also showed smad3 phosphorylation and smad2/3 nuclear translocation by Western blot and immunostaining, respectively. Moreover, with reconstituted casein and whey powder, ID1 and ID3 expression analysis was evaluated in HSC2 human oral squamous carcinoma cells. We report here that casein and whey powder caused a robust increase of TGF-β target genes interleukin11 (IL11), NADPH oxidase 4 (NOX4) and proteoglycan4 (PRG4) in gingival fibroblasts that was blocked by SB431542 and the neutralizing antibody. Moreover, casein and whey powder increased the phosphorylation of smad3 and nuclear translocation of smad2/3. No changes of proliferation markers Ki67 and cyclinD1 were observed. Furthermore, reconstituted casein and whey powder decreased ID1 and ID3 expression in the HSC2 oral squamous carcinoma cells. These findings suggest that the processing of milk into casein and whey powder maintains the TGF-β activity and its capacity to regulate ID1 and ID3 genes in oral fibroblasts and oral squamous carcinoma cells, respectively. These data increase the scientific knowledge on the biological activity of casein and whey with a special emphasis on oral health.

DIPG-59. UPREGULATION OF PRENATAL PONTINE ID1 SIGNALING IN DIPG

BACKGROUNDDiffuse intrinsic pontine gliomas (DIPGs) are lethal pediatric brain tumors with no curative therapies. Inhibitor of DNA binding (ID) proteins are key regulators of gene differentiation during embryogenesis. Previous work has shown that H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes, but this has not been validated in human DIPG, nor has the regulation and targetability of ID1 been explored in DIPG.RESULTSAnalysis of post-mortem tissue and multiple human datasets showed ID1 to be elevated in DIPG, and to correlate with reduced survival. In a multi-focal autopsy of a DIPG case, we also found ID1 expression to be heterogeneous and to correlate with tumor invasion. Chromatin immunoprecipitation qPCR (ChIP-qPCR) revealed elevated H3K27ac and low H3K27me3 at ID1 regulatory regions (enhancers/promoters) in DIPG tissue compared to normal brain, regardless of H3 or ACVR1 mutation status. Analysis of publicly-available ISH and ChIP-sequencing data of developing murine brains revealed H3K27ac at ID1 enhancers to be elevated in the prenatal hindbrain compared to prenatal forebrain and midbrain, and all postnatal brain regions. ID1 shRNA-mediated knockdown of primary human H3K27M DIPG cells (DIPG007) significantly reduced invasion and migration. We also treated DIPG007 cells with cannabidiol (CBD) and found reduced viability at clinically relevant dosing (IC50=2.4 uM) with dose-dependent reduction in ID1 protein.CONCLUSIONSThese findings indicate that a multifactorial (genetic and regionally-based) epigenetic upregulation of ID1 drives DIPG invasiveness and is targetable with CBD. ID1 knockdown and CBD treatment experiments in murine models of DIPG are ongoing.

TAMI-29. MULTIFACTORIAL UPREGULATION OF ID1 DRIVES DIPG INVASIVENESS AND IS THERAPEUTICALLY TARGETABLE

Abstract Diffuse intrinsic pontine gliomas (DIPGs) are lethal brain tumors with no effective therapies other than radiation. Inhibitor of DNA binding (ID) proteins, key regulators of lineage commitment during embryogenesis, are implicated in tumorigenesis in multiple human cancers. Prior work showed that recurrent H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes. However, this has not been validated in human DIPG. The regulation and targetability of ID1 in DIPG has not been explored either. Exome and transcriptome sequencing analysis of multi-focal DIPG tumors and normal brain tissue from autopsy (n=52) revealed that ID1 expression is significantly elevated in DIPG tissues. Higher ID1 expression correlates with reduced survival in DIPG patients and increased regional invasion in multi-focal autopsy samples. Analyses of developing mouse brain RNA/ChiP-Seq data revealed high ID1 expression and H3K27ac promoter binding in prenatal hind brain compared to all other prenatal and postnatal brain regions. ChIP-qPCR for H3K27ac and H3K27me3 revealed that ID1 gene regulatory regions are epigenetically poised for upregulation in DIPG tissues compared to normal brain, regardless of H3/ACVR1 mutational status. These data support that the developing pons is regionally poised for ID1 activation. Genetic (shRNA) ID1 knockdown in primary human H3.3K27M-DIPG cells (DIPG007) resulted in significantly reduced invasion and migration in vitro. Additionally, DIPG-ID1-KO cells showed improved sensitivity to radiation therapy. Phospho-kinase array analysis of DIPG cells revealed that Akt and WNK1 activity were significantly downregulated upon ID1 knockdown, which was previously shown in lung tumors. Treatment of DIPG007 cells with cannabidiol (CBD) reduced ID1 expression levels and viability/proliferation of DIPG cells in vitro. ID1 knockdown and CBD treatment studies in vivo are ongoing. In summary, our findings indicate that multifactorial (genetic and regional) epigenetic upregulation of ID1 drives DIPG invasiveness and targeting ID1 using CBD may be a potential strategy for the treatment of DIPGs.

Prognostic effects of the expression of inhibitor of DNA-binding family members on patients with lung adenocarcinoma.

The basic helix-loop-helix (bHLH) transcription factors are negatively regulated by inhibitor of DNA-binding (ID) proteins. Several studies have demonstrated that ID family proteins are dysregulated in a variety of cancer types, including in lung adenocarcinoma (LUAD). In current study, the prognostic value of ID family members was evaluated by investigating publicly accessible databases, including Oncomine, Kaplan-Meier plotter, UALCAN and the Human Protein Atlas. It was observed that the mRNA expression of all ID members was downregulated in LUAD tumor tissues compared with those in normal tissues according to the Oncomine and UALCAN databases. Additionally, increased mRNA expression levels of ID2 and ID1 were associated with improved and poorer survival time, respectively. Notably, ID3 and ID4 expression was not associated with survival in patients with LUAD. At the protein level, high ID2 significantly predicted an improved survival outcome while high ID1 is associated with shorter survival time. Thus, the results indicate that the ID proteins, particularly ID2, exhibit significant prognostic value in LUAD. More studies are required to elucidate the underlying molecular mechanisms behind the role of the ID family in the development of LUAD.

Early Transcriptomic Changes upon Thalidomide Exposure Influence the Later Neuronal Development in Human Embryonic Stem Cell-Derived Spheres.

Stress in early life has been linked with the development of late-life neurological disorders. Early developmental age is potentially sensitive to several environmental chemicals such as alcohol, drugs, food contaminants, or air pollutants. The recent advances using three-dimensional neural sphere cultures derived from pluripotent stem cells have provided insights into the etiology of neurological diseases and new therapeutic strategies for assessing chemical safety. In this study, we investigated the neurodevelopmental effects of exposure to thalidomide (TMD); 2,2′,4,4′-tetrabromodiphenyl ether; bisphenol A; and 4-hydroxy-2,2′,3,4′,5,5′,6-heptachlorobiphenyl using a human embryonic stem cell (hESC)-derived sphere model. We exposed each chemical to the spheres and conducted a combinational analysis of global gene expression profiling using microarray at the early stage and morphological examination of neural differentiation at the later stage to understand the molecular events underlying the development of hESC-derived spheres. Among the four chemicals, TMD exposure especially influenced the differentiation of spheres into neuronal cells. Transcriptomic analysis and functional annotation identified specific genes that are TMD-induced and associated with ERK and synaptic signaling pathways. Computational network analysis predicted that TMD induced the expression of DNA-binding protein inhibitor ID2, which plays an important role in neuronal development. These findings provide direct evidence that early transcriptomic changes during differentiation of hESCs upon exposure to TMD influence neuronal development in the later stages.

USP1 inhibitor ML323 enhances osteogenic potential of human dental pulp stem cells

LIM homeobox 8 (LHX8) is expressed during embryonic development of craniofacial tissues, including bone and teeth. In a previous study, the overexpression of LHX8 inhibited osteodifferentiation of human dental pulp stem cells (DPSCs). In this study, a cDNA microarray analysis was performed to reveal the molecular changes which occur in response to LHX8 overexpression in DPSCs and discover possible targets for an osteoinductive agent. There were 345 differentially expressed genes (DEGs) in response to osteoinductive signaling and 53 DEGs in response to LHX8 overexpression and osteoinductive signaling, respectively. Thirty-eight genes were common in both conditions, and among these, genes upregulated in LHX8 DPSCs but downregulated in osteodifferentiated DPSCs were chosen. Five of them had commercial inhibitors available. Among the tested inhibitors, ML323, which target DNA-binding protein inhibitor ID-1, promoted osteodifferentiation of DPSCs. In conclusion, inhibition of ID-1 led to increased osteogenesis of human DPSCs.

BMP4 promotes the metastasis of gastric cancer by inducing epithelial-mesenchymal transition via ID1.

Epithelial-mesenchymal transition (EMT) is a crucial process for cancer cells to acquire metastatic potential, which primarily causes death in gastric cancer (GC) patients. Bone morphogenetic protein 4 (BMP4) is a member of the TGF-β family that plays an indispensable role in human cancers. However, little is known about its roles in GC metastasis. In this study, BMP4 was found to be frequently overexpressed in GC tissues and was correlated with poor patient's prognosis. BMP4 was upregulated in GC cell lines and promoted EMT and metastasis of GC cells both in vitro and in vivo, whereas knockdown of BMP4 significantly inhibited EMT and metastasis of GC cells. Furthermore, the inhibitor of DNA binding 1 (also known as DNA-binding protein inhibitor ID1) was identified as a downstream target of BMP4 using PCR arrays and was upregulated via SMAD1/5/8 phosphorylation. ID1 knockdown attenuated BMP4-induced EMT and invasion in GC cells. Moreover, ID1 overexpression in BMP4 knockdown cells restored the promotion of EMT and cell invasion. In summary, BMP4 induced EMT and promoted GC metastasis by upregulating ID1 expression. Antagonizing BMP4 could be a potential therapeutic strategy for GC metastasis.

Carbohydrate responsive element binding protein (ChREBP) negatively regulates osteoblast differentiation via protein phosphatase 2A Cα dependent manner.

Carbohydrate responsive element binding protein (ChREBP) is a major transcription factor of lipogenesis regulated by glucose status in the liver. However, the function of ChREBP in osteogenic differentiation is unclear. The present study examined the role of ChREBP in osteoblast differentiation in MC3T3-E1 preosteoblast cell line. The mRNA expression of ChREBP, protein phosphatase 2A catalytic subunit-α (PP2A Cα) and the osteogenic genes such as, DNA-binding protein inhibitor (Id1), runt-related transcription factor-2 (Runx2), and alkaline phosphatase (ALP) was measured by qPCR and RT-PCR. Runx2, ChREBP, and PP2A Cα, protein levels were evaluated by Western blotting. ALP staining experiment was carried out to evaluate ALP enzyme activity, and a luciferase reporter assay was performed to analyze Runx2 transcriptional activity. Expression of ChREBP and PP2A Cα did not change during bone morphogenetic protein-2 (BMP2)-induced osteoblast differentiation. Overexpression of ChREBP reduced the osteogenic genes (Runx2 and ALP) expression and ALP activity, while knockdown of ChREBP had the opposite effects. Overexpression of PP2A Cα increased ChREBP expression, while inhibition of PP2A Cα using okadaic acid not only inhibited the expression of ChREBP, but also restored the mRNA and protein expression of Runx2 and activity of ALP enzyme. These results demonstrate that ChREBP inhibits BMP2-induced osteoblast differentiation in a PP2A Cα- dependent manner.

Regulation of Id2 gene expression in plasmacytoid dendritic cells

Abstract Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-I) producing cells that mediate anti-viral responses, anti-tumor immunity, and autoimmunity. When exposed to Toll-like receptor 7 (TLR7)- and TLR9-ligands, pDCs mature and produce IFN-Is; pDCs also acquire conventional DC (cDC)-like morphology and features, including the cell surface expression of antigen presentation and co-stimulatory molecules, secretion of additional cytokines, and ability to activate adaptive T cell responses. Yet, the transcriptional pathways regulating TLR-induced pDC maturation are poorly characterized. We found the mRNA and protein expression of the inhibitor of DNA binding protein 2 (Id2) was induced in TLR7/9-stimulated pDCs, and Id2 expression correlated with greater cDC-like features. Id2 normally blocks pDC development by antagonizing the pDC-master regulator, E2-2, while E2-2 suppresses Id2 transcription. Thus, we hypothesize TLR-activated signaling pathways in pDCs control the abundance of transcriptional regulators (e.g., E2-2) at the Id2 promoter as well as the promoter chromatin state, leading to induction of Id2 transcription during pDC maturation. Using genetic knockout mouse models, we found Id2 induction was independent of interferon alpha receptor and nuclear factor kappa beta signaling pathways. Chromatin-immunoprecipitation assays suggested E2-2 abundance at the Id2 promoter decreased following TLR stimulation, while activating histone modifications increased. These data are consistent with transcriptional induction of Id2 upon TLR stimulation. Future work will investigate the effect of additional regulators and TLR-regulated pathways on Id2 induction in TLR7/9-stimulated pDCs.

Milk lactoperoxidase decreases ID1 and ID3 expression in human oral squamous cell carcinoma cell lines

Milk consumption may modify the risk of squamous cell carcinoma. The role of milk to modulate the gene expression in oral squamous cell carcinoma cells has not been investigated so far. Here, HSC2 oral squamous carcinoma cells were exposed to an aqueous fraction of human milk and a whole-genome array was performed. Among the genes that were significantly reduced by human and cow milk were the DNA-binding protein inhibitor 1 (ID1), ID3 and Distal-Less Homeobox 2 (DLX2) in HSC2 cells. Also, in TR146 oral squamous carcinoma cells, there was a tendency towards a decreased gene expression. Upon size fractionation, lactoperoxidase but not lactoferrin and osteopontin was identified to reduce ID1 and ID3 in HSC2 cells. Dairy products and hypoallergenic infant formula failed to decrease the respective genes. These data suggest that milk can reduce the expression of transcription factors in oral squamous carcinoma cells.

Prognostic ability of DNA-binding protein inhibitor ID-1 expression in patients with oral squamous cell carcinoma.

DNA-binding protein inhibitor ID-1 (ID-1) plays a vital role in the development of cancer. In the present study, ID-1 expression in oral squamous cell carcinoma (OSCC), and its association with prognosis were investigated in 128 patients with OSCC, treated at the Qilu Hospital of Shandong University and followed up for an additional 10 years. Immunohistochemical analysis was performed to detect ID-1 expression, and the association between ID-1 expression and recurrence, and estimated disease-specific survival (DSS) time were subsequently analyzed using the Mann-Whitney U test and the Kaplan-Meier method, respectively. In addition, the log-rank test was implemented to compare the survival curves and multivariate Cox proportional hazards analysis was performed to assess the prognostic value of ID-1. The results demonstrated that ID-1 was highly expressed in the majority of OSCC tissues investigated, and ID-1 expression was significantly higher in cases with recurrence of local tumors and lymph node metastasis. Furthermore, higher ID-1 expression levels were associated with a shorter DSS time. Taken together, the results of the present study suggest that ID-1 may serve as an independent prognostic factor to predict DSS time in patients with OSCC.

Effects of Monoterpenes of Trachyspermum ammi on the Viability of Spermatogonia Stem Cells In Vitro.

Trachyspermum ammi (Apiaceae) plants have several medicinal and condimentary applications and are considered an aphrodisiac agent in Iranian Traditional Medicine. Thus, the present study aims to evaluate the effects of oil from Iranian T. ammi plants on the viability of spermatogonial stem cells in vitro. The essential oil of T. ammi fruits was extracted by hydrodistillation, and the amount of thymol was calculated by a gas-chromatography method. Spermatogonial stem cells were isolated from the testes of mice using enzyme digestion. Real-time polymerase chain reaction (RT-PCR) was applied to assess the gene expressions of promyelocytic leukemia zinc finger protein (Plzf), DNA-binding protein inhibitor (ID-4), tyrosine-protein kinase (c-Kit), B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (BAX). The number and diameter of colonies were also measured in the treated cells. The amount of thymol in the oil was 130.7 ± 7.6 µg/mL. Flow cytometry analysis showed that 92.8% of all cells expressed stimulated by retinoic acid 8 (Stra8), a spermatogonial stem cell marker. Expression of Plzf and ID-4 genes significantly increased in the treatment groups, while c-Kit and BAX decreased, and Bcl2 increased in the presence of essential oil. The numbers and diameters of cells were also improved by the application of the plant oil. These data indicated that monoterpenes from the oil of T. ammi improved the quality and viability of spermatogonia cells in the cell culture.

Inhibitor of Differentiation 1 (Id1) in Cancer and Cancer Therapy.

The inhibitor of DNA binding (Id) proteins are regulators of cell cycle and cell differentiation. Of all Id family proteins, Id1 is mostly linked to tumorigenesis, cellular senescence as well as cell proliferation and survival. Id1 is a stem cell-like gene more than a classical oncogene. Id1 is overexpressed in numerous types of cancers and exerts its promotion effect to these tumors through different pathways. Briefly, Id1 was found significantly correlated with EMT-related proteins, K-Ras signaling, EGFR signaling, BMP signaling, PI3K/Akt signaling, WNT and SHH signaling, c-Myc signaling, STAT3 signaling, RK1/2 MAPK/Egr1 pathway and TGF-β pathway, etc. Id1 has potent effect on facilitating tumorous angiogenesis and metastasis. Moreover, high expression of Id1 plays a facilitating role in the development of drug resistance, including chemoresistance, radiation resistance and resistance to drugs targeting angiogenesis. However, controversial results were also obtained. Overall, Id1 represent a promising target of anti-tumor therapeutics based on its potent promotion effect to cancer. Numerous drugs were found exerting their anti-tumor function through Id1-related signaling pathways, such as fucoidan, berberine, tetramethylpyrazine, crizotinib, cannabidiol and vinblastine.