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The incidence and mortality of colorectal cancer (CRC) are ranked in the top three worldwide in 2020. Abundant studies have reported that circular RNAs (circRNAs) act critical roles in the genesis and development of tumors, including CRC. Nevertheless, the roles and detailed regulation mechanisms of circRNAs that are related to the initiation and development of CRC have not been fully found and clarified. This research primarily revealed that circTMEM59 was greatly downregulated in CRC tissues and cell lines via qRT-PCR. In addition, the decreased expression of circTMEM59 was closely related to adverse clinicopathological characteristics and the shorter survival time of CRC patients. Then, a further study found that the overexpression of circTMEM59 suppressed cell growth and accelerated the cell death of CRC via a series of experiments in vitro and in vivo. Furthermore, circTMEM59 also repressed the metastatic behaviors of CRC cells. Further study revealed that circTMEM59 played the role of competing endogenous RNAs (ceRNAs) by binding to miR-668-3p to increase the expression of inhibitor of DNA binding 4 (ID4) in CRC. In summary, the results of this study clarified the antitumor effects of circTMEM59/miR-668-3p/ID4 axis in CRC progression and provided potential therapeutic targets and clinical prognostic markers for CRC.

Id3, an inhibitor of DNA binding protein, plays important roles in the function and homeostasis of effector and memory T cells. Recent evidence has shown that Id3 is also implicated in CD8 T cell exhaustion. However, whether and how Id3 might regulate effector function or exhaustion of CD8 T cells, especially in the tumor setting, is still unknown. Here, we first showed that Id3 expression was impaired in tumor-infiltrating CD8 T cells as liver cancer progressed, especially in PD-1 +Tim-3 + exhausted CD8 T cells. Enforced expression of Id3 in CD8 T cells resulted in repressed development of anti-tumor CTLs exhaustion, which offered better tumor control. And partially depletion of Id3 in CD8 T cells promoted the development of exhausted CD8 T cells. Furthermore, Id3hi CD8 T cells could respond to PD-1 blockade. Collectively, Id3 exerts protective functions in CD8 T cells for liver cancer.

Background During pregnancy, trophoblast cell invasion needs to be finely controlled. Aberrant trophoblast cell invasion is associated with placental diseases. Epidermal growth factor (EGF) and its receptor, EGFR, are expressed in trophoblast cells. Although the pro-invasive effect of EGF on trophoblast cells has been reported, the underlying mechanism remains largely unknown. Results In the present study, we conducted an RNA sequencing (RNA-seq) to HTR-8/SVneo human trophoblast cells in response to EGF and identified KISS1 as a target gene of EGF. The human KISS1 gene encodes kisspeptin, also known as metastin, which can suppress tumor metastasis. Our results showed that EGF treatment downregulated KISS1 expression and secretion by activating the EGFR-mediated PI3K/AKT signaling pathway. In addition, the expression of inhibitor of DNA-binding protein 3 (ID3) was downregulated by EGF and that was required for the EGF-suppressed KISS1 expression. Functionally, transwell invasion assays demonstrated that EGF stimulated human trophoblast cell invasion by downregulating KISS1 expression. Preeclampsia (PE) is a placental disease characterized by insufficient trophoblast cell invasion. Our clinical results revealed that serum levels of EGF were downregulated while serum and placental levels of KISS1 were upregulated in PE patients. Conclusions This study demonstrates that downregulation of EGF can lead to poor trophoblast cell invasion by increasing KISS1 expression which subsequently contributes to the pathogenesis of PE.

The inhibitor of DNA-binding 3 (ID3) is a transcriptional regulator that limits interaction of basic helix-loop-helix transcription factors with their target DNA sequences. We previously reported that ID3 loss is associated with mutational signatures linked to DNA repair defects. Here we demonstrate that ID3 exhibits a dual role to promote DNA double-strand break (DSB) repair, particularly homologous recombination (HR). ID3 interacts with the MRN complex and RECQL helicase to activate DSB repair and it facilitates RAD51 loading and downstream steps of HR. In addition, ID3 promotes the expression of HR genes in response to ionizing radiation by regulating both chromatin accessibility and activity of the transcription factor E2F1. Consistently, analyses of TCGA cancer patient data demonstrate that low ID3 expression is associated with impaired HR. The loss of ID3 leads to sensitivity of tumor cells to PARP inhibition, offering new therapeutic opportunities in ID3-deficient tumors., Graphical Abstract Graphical AbstractDual role of ID3 in promoting homologous recombination.

Introduction The 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. Methods We 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. Results We 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. Discussion This 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.

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.

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.

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.

Memory CD4

Background The importance of spermatogonial stem cells (SSCs) in spermatogenesis is crucial and intrinsic factors and extrinsic signals mediate fate decisions of SSCs. Among endogenous regulators, microRNAs (miRNAs) play critical role in spermatogenesis. However, the mechanisms which individual miRNAs regulate self- renewal and differentiation of SSCs are unknown. The aim of this study was to investigate effects of miRNA-30a-5p inhibitor on fate determinations of SSCs. Methods SSCs were isolated from testes of neonate mice (3–6 days old) and their purities were performed by flow cytometry with ID4 and Thy1 markers. Cultured cells were transfected with miRNA- 30a-5p inhibitor. Evaluation of the proliferation (GFRA1, PLZF and ID4) and differentiation (C-Kit & STRA8) markers of SSCs were accomplished by immunocytochemistry and western blot 48 h after transfection. Results Based on the results of flow cytometry with ID4 and Thy1 markers, percentage of purity of SSCs was about 84.3 and 97.4 % respectively. It was found that expression of differentiation markers after transfection was significantly higher in miRNA-30a- 5p inhibitor group compared to other groups. The results of proliferation markers evaluation also showed decrease of GFRA1, PLZF and ID4 protein in SSCs transfected with miRNA-30a-5p inhibitor compared to the other groups. Conclusions It can be concluded that inhibition of miRNA-30a-5p by overexpression of differentiation markers promotes differentiation of Spermatogonial Stem Cells.

Aim: The mechanistic role of inhibitor of DNA binding or differentiation (ID) family in ovarian cancer (OC) has remained unclear. Materials & methods: We used the Oncomine, GEPIA, Kaplan–Meier Plotter, cBioPortal, SurvExpress, PROGgene V2, TIMER, and FunRich to evaluate the prognostic value of IDs in patients with OC. Results: the mRNA transcripts of all IDs were markedly downregulated in OC compared with normal tissue. The prognostic value of IDs was also explored within the subtypes, pathological stages, clinical stages and TP53 mutational status. The group with low-risk IDs showed relatively good overall survival (OS) compared with the high-risk group. Conclusion: ID1/3/4 may be exploited as promising prognostic biomarkers and therapeutic targets in OC patients.

Medulloblastoma (MB), the most common malignant childhood brain tumor, is a serious threat to life. Circular RNA (circRNA) is involved in the development of various cancers, including MB. We aimed to explore the role of circRNA spindle and kinetochore associated complex subunit 3 (circ-SKA3) in MB progression. Circ-SKA3 expression was elevated in MB tissues and cells. Depleted expression of circ-SKA3 inhibited MB cell proliferation, migration and invasion and induced apoptosis and cell cycle arrest, and circ-SKA3 knockdown inhibited MB growth in vivo. Mechanism analyses revealed that circ-SKA3 directly targeted miR-326 that could bind to ID3, and circ-SKA3 decoyed miR-326 to increasing ID3 expression. Rescue experiments showed that miR-326 inhibition reversed the effects of circ-SKA3 knockdown, and ID3 overexpression recovered MB cell proliferation, migration and invasion blocked by miR-326 restoration. In conclusion, circ-SKA3 functioned as an oncogene to promote the development of MB by increasing ID3 expression via decoying miR-326, hinting that circ-SKA3 might be a therapeutic target of MB.

T cells develop in the thymus from lymphoid primed multipotent progenitors or common lymphoid progenitors into αβ 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.

As the clinical failure of glioblastoma treatment is attributed by multiple components, including myelin-associated infiltration, assessment of the molecular mechanisms underlying such process and identification of the infiltrating cells have been the primary objectives in glioblastoma research. Here, we adopted radiogenomic analysis to screen for functionally relevant genes that orchestrate the process of glioma cell infiltration through myelin and promote glioblastoma aggressiveness. The receptor of the Nogo ligand (NgR1) was selected as the top candidate through Differentially Expressed Genes (DEG) and Gene Ontology (GO) enrichment analysis. Gain and loss of function studies on NgR1 elucidated its underlying molecular importance in suppressing myelin-associated infiltration in vitro and in vivo. The migratory ability of glioblastoma cells on myelin is reversibly modulated by NgR1 during differentiation and dedifferentiation process through deubiquitinating activity of USP1, which inhibits the degradation of ID1 to downregulate NgR1 expression. Furthermore, pimozide, a well-known antipsychotic drug, upregulates NgR1 by post-translational targeting of USP1, which sensitizes glioma stem cells to myelin inhibition and suppresses myelin-associated infiltration in vivo. In primary human glioblastoma, downregulation of NgR1 expression is associated with highly infiltrative characteristics and poor survival. Together, our findings reveal that loss of NgR1 drives myelin-associated infiltration of glioblastoma and suggest that novel therapeutic strategies aimed at reactivating expression of NgR1 will improve the clinical outcome of glioblastoma patients.

Supplemental Digital Content is available in the text., Objective: Pulmonary arterial hypertension is a disease of proliferative vascular occlusion that is strongly linked to mutations in BMPR2—the gene encoding the BMPR-II (BMP [bone morphogenetic protein] type II receptor). The endothelial-selective BMPR-II ligand, BMP9, reverses disease in animal models of pulmonary arterial hypertension and suppresses the proliferation of healthy endothelial cells. However, the impact of BMPR2 loss on the antiproliferative actions of BMP9 has yet to be assessed. Approach and Results: BMP9 suppressed proliferation in blood outgrowth endothelial cells from healthy control subjects but increased proliferation in blood outgrowth endothelial cells from pulmonary arterial hypertension patients with BMPR2 mutations. This shift from growth suppression to enhanced proliferation was recapitulated in control human pulmonary artery endothelial cells following siRNA-mediated BMPR2 silencing, as well as in mouse pulmonary endothelial cells isolated from endothelial-conditional Bmpr2 knockout mice (Bmpr2EC−/−). BMP9-induced proliferation was not attributable to altered metabolic activity or elevated TGFβ (transforming growth factor beta) signaling but was linked to the prolonged induction of the canonical BMP target ID1 in the context of BMPR2 loss. In vivo, daily BMP9 administration to neonatal mice impaired both retinal and lung vascular patterning in control mice (Bmpr2EC+/+) but had no measurable effect on mice bearing a heterozygous endothelial Bmpr2 deletion (Bmpr2EC+/−) and caused excessive angiogenesis in both vascular beds for Bmpr2EC−/− mice. Conclusions: BMPR2 loss reverses the endothelial response to BMP9, causing enhanced proliferation. This finding has potential implications for the proposed translation of BMP9 as a treatment for pulmonary arterial hypertension and suggests the need for focused patient selection in clinical trials. Graphic Abstract: A graphic abstract is available for this article.

Treatment options for brain metastatic breast cancer are limited because the molecular mechanism for how breast cancer cells infiltrate the brain is not fully understood. For breast tumors to metastasize to the brain first, cells need to detach from the primary tumor, enter in the blood circulation, survive within the microvascular niche, and then cross the blood-brain barrier (BBB) to colonize into the brain. It is critical to understand how breast cancer cells transmigrate through the BBB to prevent brain metastasis. Nuclear respiratory factor 1 (NRF1) transcription factor has been reported to be highly active in several human cancers and its aberrant expression facilitates in the acquisition of breast cancer stem cells (BCSCs). Inhibitor of differentiation protein 3 (ID3), a transcription regulating protein, induces pluripotent endothelial stem cells (ESCs). Herein, we investigated if NRF1-induced BCSCs could cross a BBB model and guiding of BCSCs by ID3-induced ESCs across the BBB. BCSCs and ESCs were subjected to functional gain/loss experiments to determine if NRF1/ID3 contributed to lineage-specific BCSCs organ entry. First, we tested whether NRF1 promoted migration of breast cancer using a BBB model consisting of BCSCs or MDA-MB231 cells, brain endothelial cell layer, and astrocytes. NRF1 overexpression increased the propensity for BCSCs and NRF1-induced MDA-MB231 cells to adhere to brain endothelial cells and migrate across a human BBB model. Increased adhesion of NRF1-induced BCSCs to ESCs

Although mechanical forces are involved in pressure-overloaded cardiomyopathy, their effects on gene transcription profiles are not fully understood. Here, we used next-generation sequencing (NGS) to investigate changes in genomic profiles after cyclic mechanical stretching of human cardiomyocytes. We found that 85, 87, 32, 29, and 28 genes were differentially expressed after 1, 4, 12, 24, and 48 hours of stretching. Furthermore, 10 of the 29 genes that were up-regulated and 11 of the 28 that were down-regulated after 24 h showed the same changes after 48 h. We then examined expression of the genes that encode serpin family E member 1 (SERPINE1), DNA-binding protein inhibitor 1 (ID1), DNA-binding protein inhibitor 3 (ID3), and CCL2, a cytokine that acts as chemotactic factor in monocytes, in an RT-PCR experiment. The same changes were observed for all four genes after all cyclic stretching durations, confirming the NGS results. Taken together, these findings suggest that cyclical stretching can alter cardiac cell physiology by activating cardiac cell metabolism and impacting cholesterol biosynthesis signaling.

microRNAs may function as oncogenes or tumor suppressor genes that play crucial roles in human carcinogenesis and cancer development. Growing evidence revealed that the tumor suppressor Id3 is involved in tumor progression, carcinogenesis, and the tumor microenvironment. We identified miR-212-5p as a negative posttranscriptional modulator of Id3. Dual luciferase reporter assay was used to verify that Id3 is a direct target gene of miR-212-5p. Id3 was lowly expressed and miR-212-5p was highly expressed in non-small-cell lung cancer (NSCLC) tissues and cells. In addition, we found that NSCLC patients having a higher level of miR-212-5p expression had a shorter survival time. Besides this, miR-212-5p could directly target Id3 and reduce its expression. miR-212-5p overexpression significantly accelerated cell proliferation, migration, and invasion by reversing the effects of Id3. Id3 overexpression by silencing miR-212-5p expression suppressed phosphatidylinositol 3 kinase (PI3K)/Akt activity and consequently promoted apoptosis and inhibited cell proliferation in lung cancer cells. Consistent with the in vitro results, a xenograft mouse model was used to validate the fact that miR-212-5p could promote tumorigenesis by targeting Id3 and activate the PI3K/Akt pathway in vivo as well. Taken together, the present results indicated that miR-212-5p may be involved in progression of NSCLC through the PI3K/Akt signaling pathway by targeting Id3.

Aim This study aimed to explore the miR-342-3p expression in pre-eclampsia (PE) placentas and confirm whether miR-342-3p exerts effects on proliferation and migration of HTR-8/SVneo trophoblastic cells. Methods The PE placentas (n = 8) were taken from gravidas complicated by PE and delivered after 34 weeks. The chorionic plates and the basal plates were separately taken from the placenta disc near the position of umbilical cord insertion. RT-qPCR was used to measure the expression of miR-342-3p in the chorionic plates and the basal plates. Cell invasion assay and MMT assay were used to assess the effects of miR-342-3p on proliferation and migration of HTR-8/SVneo trophoblastic cells. Luciferase reporter assay and Western blotting were used to analyze the target of miR-342-3p and investigate the detailed mechanisms. Results The expression of miR-342-3p was upregulated in both basal plates and chorionic plates in patients with PE compared with healthy pregnant individuals. MiR-342-3p inhibitor suppressed the cell viability and invasion, and induced apoptosis in trophoblast cells. Furthermore, inhibitor of DNA binding (ID)-4 (ID4) was a direct target of miR-342-3p, and knockdown of ID4 abrogated the regulation effect of miR-342-3p on cell viability, apoptosis and invasion. Conclusion Inhibition of miR-342-3p expression may suppress the occurrence of PE by targeting ID4 in vitro.

OBJECTIVE: Neovascularization can maintain and even improve tissue perfusion in the setting of limb ischemia during peripheral artery disease (PAD). The molecular and cellular mechanisms mediating this process are incompletely understood. We investigate the potential role(s) for Inhibitor of differentiation 3 (Id3) in regulating blood flow in a murine model of hind limb ischemia (HLI). APPROACH AND RESULTS: HLI was modeled through femoral artery ligation and resection and blood flow recovery was quantified by Laser Doppler perfusion imaging. Mice with global Id3 deletion had significantly impaired perfusion recovery at 14 and 21 days of HLI. Endothelial- or myeloid cell-specific deletion of Id3 revealed no effect on perfusion recovery while B cell-specific knockout of Id3 (Id3(BKO)) revealed a significant attenuation of perfusion recovery. Flow cytometry revealed no differences in ischemia-induced T cells or myeloid cell numbers at 7 days of HLI, yet there was a significant increase in B-1b cells in Id3(BKO). Consistent with these findings, ELISA demonstrated increases in skeletal muscle and plasma IgM. In vitro experiments demonstrated reduced proliferation and increased cell death when ECs were treated with conditioned media from IgM-producing B-1b cells and tibialis anterior muscles in Id3(BKO) mice showed reduced density of total CD31(+) and αSMA(+)CD31(+) vessels. CONCLUSIONS: This study is the first to demonstrate a role for B cell-specific Id3 in maintaining blood flow recovery during HLI. Results suggest a role for Id3 in promoting blood flow during HLI and limiting IgM-expressing B-1b cell expansion. These findings present new mechanisms to investigate in PAD pathogenesis.

The mesenchymal stem cell-derived exosome (MSCs-exo) carrying microRNAs have been proved to regulate tumor biological activities. Clarifying molecular mechanism and identifying predictive microRNAs will be of great value in anti-tumor therapy improvement.We aimed to investigate the regulatory role of microRNA-342-3p (miR-342-3p) in MSCs-exo on breast cancer.Breast cancer tissues and cell lines were used to evaluate miR-342-3p expression in patients with or without lymph node/distal organ metastasis. The impact of MSCs-exo expression on tumor cell chemo-resistance and invasion/migration was measured. Dual-luciferase reporter gene assay was applied to identify binding site. Inhibitor of differentiation 4 (ID4) siRNA and miR-342-3p inhibitor transfection was conducted to further explore the miR-342-3p/ID4 axis on chemo-resistance and metastasis of breast cancer cells.Breast cancer cells revealed significantly lower level of miR-342-3p in patients with metastatic diseases. miR-342-3p suppressed invasive and chemo-resistant behavior of breast cancer tumor cells. Binding site between miR-342-3p and ID4 was proved. ID4 could reverse the influence of miR-342-3p on chemo-resistance. The tumor inhibition effect of IDA siRNA in vivo was also identified.This study demonstrated that miR-342-3p acted as potential tumor suppressor by inhibiting metastasis and chemo-resistance of breast cancer cells through targeting ID4. This study might provide potential therapy targets for the treatment of breast cancer.

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.

Objectives Evidence reveals that microRNAs (miRNAs) are abnormally expressed in lung adenocarcinoma (LUAD) tissue and are crucial in LUAD occurrence. Therefore, this study aims to find the miRNA which could regulate LUAD and to further explore its regulatory mechanism, thus providing a potential molecular target for LUAD. Methods miR-9-5p and ID4 expression in LUAD cells were measured by real-time quantitative PCR and western blot. Cell functional assays were conducted to detect the biological functions of LUAD cells. A dual-luciferase reporter assay was utilized to validate the binding relationship between miR-9-5p and ID4. Results miR-9-5p was highly expressed whereas ID4 was lowly expressed in LUAD. miR-9-5p facilitated LUAD cell progression by targeting ID4. Conclusion miR-9-5p promotes LUAD cell progression by modulating ID4 and may become a potential target for LUAD.

Pancreatic cancer (PC) is the seventh-leading cause of cancer-related mortality, and is associated with limited therapeutic options and poor prognosis. The extracellular matrix (ECM) represents the main component of the tumor microenvironment. Studies have found controversial roles of osteoglycin (OGN), a classical small leucine-rich proteoglycan found in the ECM in human malignancies; however, the significance of OGN in PC has not been determined. Here, the expression profiles of OGN in PC tissues and cell lines were evaluated by Gene Expression Profiling Interactive Analysis (GEPIA) database, immunohistochemistry, western blot, and quantitative PCR. OGN was found to be significantly upregulated in PC tissues and cell lines. Moreover, the expression of OGN was observed to be closely associated with TNM stage, stage III showed a higher OGN expression than that of stages I and II. Survival analysis showed that patients with PC showing high levels of OGN had low survival rates. The effects of OGN on cell proliferation and apoptosis were analyzed using MTT, CCK8, EdU and TUNEL assays. Wound-healing and invasion assays were conducted to test migratory and invasive abilities. Overexpression of OGN was demonstrated to promote proliferation, migration, and invasion, and inhibit apoptosis of PC cells. Further experiments revealed that inhibitor of DNA binding 4 (ID4) was upregulated by OGN. Silencing ID4 by small interfering RNA was shown to partially reverse the tumor-promoting effect of OGN. Collectively, our preliminary results indicate that the elevated expression of OGN may be associated with PC progression and may serve as a potential biomarker for the diagnosis and prognosis of PC. Targeting of OGN/ID4 axis may be a promising strategy in PC therapy.

The differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes is the prerequisite for remyelination in demyelinated disorders such as multiple sclerosis (MS). Epigenetic mechanisms, such as DNA methylation, have been suggested to control the intricate network of transcription factors involved in OPC differentiation. Yet, the exact mechanism remains undisclosed. Here, we are the first to identify the DNA-binding protein inhibitors, Id2 and Id4, as targets of DNA methylation during OPC differentiation. Using state-of-the-art epigenetic editing via CRISPR/dCas9-DNMT3a, we confirm that targeted methylation of Id2/Id4 drives OPC differentiation. Moreover, we show that in the pathological context of MS, methylation and gene expression levels of both ID2 and ID4 are altered compared to control human brain samples. We conclude that DNA methylation is crucial to suppress ID2 and ID4 during OPC differentiation, a process that appears to be dysregulated during MS. Our data do not only reveal new insights into oligodendrocyte biology, but could also lead to a better understanding of CNS myelin disorders.

Inhibitor of DNA binding (Id) is a dominant negative form of the E-box binding basic-helix-loop-helix (bHLH) transcription factor since it is devoid of the basic region required for DNA binding and forms an inactive hetero dimer with bHLH proteins. The E-box sequence located in the promoter region of the GATA-binding protein 4 (GATA-4) gene is essential for transcriptional activation in P19CL6 cells. These cells differentiate into cardiomyocytes and start to express GATA-4, which further triggers cardiac-specific gene expression. In this study, expression plasmids for Ids tagged with human influenza hemagglutinin (HA)-FLAG were constructed and introduced into P19CL6 cells. The stable clones expressing the recombinant Id proteins (Id1 or Id3) were isolated. The GATA-4 gene expression in these clones under differentiation condition in the presence of 1% dimethyl sulfoxide (DMSO) was repressed, with concomitant abolishment of the transcription of α-myosin heavy chain (α-MHC), which is a component of cardiac myofibrils. Thus, the increased expression of Id protein could affect GATA-4 gene expression and negatively regulate the differentiation of P19CL6 cells.

Inhibitor of DNA binding (Id) genes comprise a family of four helix–loop–helix (HLH) transcriptional inhibitors. Our earlier studies revealed a role for ID2 within the circadian system, contributing to input, output, and core clock function through its interaction with CLOCK and BMAL1. Here, we explore the contribution of ID4 to the circadian system using a targeted disruption of the Id4 gene. Attributes of the circadian clock were assessed by monitoring the locomotor activity of Id4−/− mice, and they revealed disturbances in its operation. Id4-mutant mice expressed a shorter circadian period length, attenuated phase shifts in responses to continuous and discrete photic cues, and an advanced phase angle of entrainment under a 12:12 light:dark cycle and under short and long photoperiods. To understand the basis for these properties, suprachiasmatic nucleus (SCN) and retinal structures were examined. Anatomical analysis reveals a smaller Id4−/− SCN in the width dimension, which is a finding consistent with its smaller brain. As a result of this feature, anterograde tracing in Id4−/− mice revealed retinal afferents innovate a disproportionally larger SCN area. The Id4−/− photic entrainment responses are unlikely to be due to an impaired function of the retinal pathways since Id4−/− retinal anatomy and function tested by pupillometry were similar to wild-type mice. Furthermore, these circadian characteristics are opposite to those exhibited by the Id2−/− mouse, suggesting an opposing influence of the ID4 protein within the circadian system; or, the absence of ID4 results in changes in the expression or activity of other members of the Id gene family. Expression analysis of the Id genes within the Id4−/− SCN revealed a time-of-day specific elevated Id1. It is plausible that the increased Id1 and/or absence of ID4 result in changes in interactions with bHLH canonical clock components or with targets upstream and/or downstream of the clock, thereby resulting in abnormal properties of the circadian clock and its entrainment.

Cutaneous wound healing requires intricate synchronization of several key processes. Among them, local nerve regeneration is known to be vitally important for proper repair. However, the underlying mechanisms of local nerve regeneration are still unclear. Fibroblasts are one of the key cell types within the skin whose role in local nerve regeneration has not been extensively studied. In our study, we found skin fibroblasts were in tight contact with regenerated nerves during wound healing, while rare interactions were shown under normal circumstances. Moreover, skin fibroblasts surrounding the nerves were shown to be activated and reprogrammed to exhibit neural cell‐like properties by upregulated expressing inhibitor of DNA binding 1 (ID1) and ID3. Furthermore, we identified the regulation of integrin α6 (Itga6) by ID1/ID3 in fibroblasts as the mechanism for axon guidance. Accordingly, transplantation of the ID1/ID3‐overexpressing fibroblasts or topical injection of ID1/ID3 lentivirus significantly promoted local nerve regeneration and wound healing following skin excision or sciatic nerve injury. Therefore, we demonstrated a new role for skin fibroblasts in nerve regeneration following local injury by directly contacting and guiding axon regrowth, which might hold therapeutic potential in peripheral nerve disorders and peripheral neuropathies in relatively chronic refractory wounds., Skin fibroblasts contribute to local nerve regeneration by directly guiding axon regrowth, which suggests a potential therapeutic strategy for peripheral nerve disorders and relatively chronic refractory wounds.

Rhabdomyosarcoma (RMS), is the most frequent soft tissue tumor in children that originates from disturbances in differentiation process. Mechanisms leading to the development of RMS are still poorly understood. Therefore, by analysis of two RMS RH30 cell line subclones, one subclone PAX7 negative, while the second one PAX7 positive, and comparison with other RMS cell lines we aimed at identifying new mechanisms crucial for RMS progression. RH30 subclones were characterized by the same STR profile, but different morphology, rate of proliferation, migration activity and chemotactic abilities in vitro, as well as differences in tumor morphology and growth in vivo. Our analysis indicated a different level of expression of adhesion molecules (e.g., from VLA and ICAM families), myogenic microRNAs, such as miR-206 and transcription factors, such as MYOD, MYOG, SIX1, and ID. Silencing of PAX7 transcription factor with siRNA confirmed the crucial role of PAX7 transcription factor in proliferation, differentiation and migration of RMS cells. To conclude, our results suggest that tumor cell lines with the same STR profile can produce subclones that differ in many features and indicate crucial roles of PAX7 and ID proteins in the development of RMS.

Stage- and cell type-specific biomarkers are important for understanding spermatogenesis in mammalian testis. The present study identified several testicular cell marker proteins in 6- and 24-month old bovine testes. In 6-month old bovine testes, spermatogonia and spermatocytes were detected but complete spermatogenesis occurred in 24-month old testes. The diameters of the seminiferous tubules increased significantly in the 24-month old testes compared with those in the 6-month old testes. Protein Gene Product 9.5 (PGP9.5), also known as the undifferentiated spermatogonium marker, and GATA4 (GATA binding protein 4), vimentin, and SOX9 (SRY-Box Transcription Factor 9) were detected in the basement membrane region. Interestingly, ID4 (inhibitor of DNA binding protein 4; previously known as the undifferentiated cell marker) proteins were located in the basement membrane region but their expression patterns were different from those of PGP9.5. Co-immunohistochemistry results showed that ID4 was detected in the Sertoli cells expressing vimentin and SOX9 in 6- and 24-month old bovine testes. This result indicated that ID4 is a putative biomarker of Sertoli cell in the bovine system, which is different from the rodent models. Thus, these results will contribute in understanding the process of spermatogenesis that is different in bovines compared to other species.

Iron overload promotes the generation of reactive oxygen species (ROS). Pancreatic β-cells can counter oxidative stress through multiple anti-oxidant responses. Herein, RNA-sequencing was used to describe the expression profile of iron regulatory genes in human islets with or without diabetes. Functional experiments including siRNA silencing, qPCR, western blotting, cell viability, ELISA and RNA-sequencing were performed as means of identifying the genetic signature of the protective response following iron overload-induced stress in human islets and INS-1. FTH1 and FTL genes were highly expressed in human islets and INS-1 cells, while hepcidin (HAMP) was low. FXN, DMT1 and FTHL1 genes were differentially expressed in diabetic islets compared to control. Silencing of Hamp in INS-1 cells impaired insulin secretion and influenced the expression of β-cell key genes. RNA-sequencing analysis in iron overloaded INS-1 cells identified Id1 and Id3 as the top down-regulated genes, while Hmox1 was the top upregulated. Expression of ID1, ID3 and HMOX1 was validated at the protein level in INS-1 cells and human islets. Differentially expressed genes (DEGs) were enriched for TGF-β, regulating stem cells, ferroptosis, and HIF-1 signaling. Hmox1-silenced cells treated with FAC elevated the expression of Id1 and Id3 expression than untreated cells. Our findings suggest that HMOX1, ID1 and ID3 define the response mechanism against iron-overload-induced stress in β-cells.

Foot-and-mouth disease virus (FMDV) is an important pathogen that harms cloven-hoofed animals and has caused serious losses to livestock production since its discovery. Furthermore, inhibitor of DNA binding (ID) proteins have been thoroughly studied in tumorigenesis, differentiation and metastasis, but its role in viral infection is rarely known. In this study, three gene knockout cell lines ID1 KO, ID3 KO, ID1/3 KO were obtained based on BHK-21 cells. We found that ID1 and ID3 genes single or double knockout promote the replication of FMDV. Moreover, compared with negative control cells during virus infection, there were 551 up-regulated genes and 1222 down-regulated genes in the ID1 KO cell line; 916 up-regulated genes and 1845 down-regulated genes in the ID3 KO cell line; 810 up-regulated genes and 1566 down-regulated genes in ID1/3 KO cell line. Further genes expression patterns verification results also showed a good correlation between the data of RT-qRCR and RNA-seq. These findings provide a basis for studying the relevant mechanisms between host genes and ID genes during FMDV infection.

CD8+ memory T (TM) cells play a critical role in immune defense against infection. Two common γ-chain family cytokines, IL-2 and IL-7, although triggering the same mTORC1–S6K pathway, distinctly induce effector T (TE) cells and TM cells, respectively, but the underlying mechanism(s) remains elusive. In this study, we generated IL-7R-/and AMPKα1-knockout (KO)/OTI mice. By using genetic and pharmaceutical tools, we demonstrate that IL-7 deficiency represses expression of FOXO1, TCF1, p-AMPKα1 (T172), and p-ULK1 (S555) and abolishes T cell memory differentiation in IL-7R KO T cells after Listeria monocytogenesis rLmOVA infection. IL-2– and IL-7–stimulated strong and weak S6K (IL-2/S6Kstrong and IL-7/S6Kweak) signals control short-lived IL-7R−CD62L−KLRG1+ TE and long-term IL-7R+CD62L+KLRG1− TM cell formations, respectively. To assess underlying molecular pathway(s), we performed flow cytometry, Western blotting, confocal microscopy, and Seahorse assay analyses by using the IL-7/S6Kweak–stimulated TM (IL-7/TM) and the control IL-2/S6Kstrong–stimulated TE (IL-2/TE) cells. We determine that the IL-7/S6Kweak signal activates transcriptional FOXO1, TCF1, and Id3 and metabolic p-AMPKα1, p-ULK1, and ATG7 molecules in IL-7/TM cells. IL-7/TM cells upregulate IL-7R and CD62L, promote mitochondria biogenesis and fatty acid oxidation metabolism, and show long-term cell survival and functional recall responses. Interestingly, AMPKα1 deficiency abolishes the AMPKα1 but maintains the FOXO1 pathway and induces a metabolic switch from fatty acid oxidation to glycolysis in AMPKα1 KO IL-7/TM cells, leading to loss of cell survival and recall responses. Taken together, our data demonstrate that IL-7–stimulated weak strength of mTORC1–S6K signaling controls T cell memory via activation of transcriptional FOXO1–TCF1–Id3 and metabolic AMPKα1–ULK1–ATG7 pathways. This (to our knowledge) novel finding provides a new mechanism for a distinct IL-2/IL-7 stimulation model in T cell memory and greatly impacts vaccine development.

Purpose In 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. Methods IOP 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. Results Over-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. Conclusions Increased 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.

Triple-negative (TN) breast cancer represents a subtype of breast cancer that does not express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER-2). Clinically, it is characterized by high invasiveness, high metastatic potential, and poor prognosis. Inhibitor of DNA binding 4 (ID4) has been shown to be overexpressed in these tumors acting as an oncogene responsible for many of its aggressive features. CDC42, a plasma membrane-associated small GTPase, can downregulate ID4 gene expression through hypermethylation of its promoter in colorectal adenocarcinomas. Since ID4 acts as an oncogene and is hypomethylated in TN breast tumors, here we asked whether CDC42 could also epigenetically silence ID4 and in doing so revert aggressive features of this tumor type.Gene expression was retrieved from TCGA database using UCSC Xena. Association between overall survival (OS) and gene expression was assessed using Kaplan-Meier plotter. In vitro experiments involved ectopic expression of CDC42 in MDA-MB231and in MDA-MB468 breast cancer cell lines. Gene expression was analyzed by qPCR, western blot and inmunofluorescence assays and methylation by MSP, MS-MLPA, or ddMSP.Data mining analysis revealed that CDC42 expression varies among breast cancer subtypes that in the basal-like subtype there is an inverse correlation between CDC42 and ID4 expression and a positive correlation between CDC42 expression and ID4 methylation. In vitro experiments revealed that CDC42 overexpression induced ID4 methylation through the activation of the EZH2 pathway. ID4 silencing produced an increase in BRCA1 expression and a less aggressive phenotype in the tested cell line.We show that CDC42 silences ID4 through methylation in TN breast cancer. Given that ID4 acts as an oncogene in these tumors, we think that finding an epigenetic regulator of ID4 contributes to the research and clinical management of TN breast tumors.

T cells are essential mediators of the immune responses against infectious diseases and provide long-lived protection from reinfection. The differentiation of naive T cells to effector T cells and subsequent differentiation and persistence of memory T cell populations in response to infection is a highly regulated process. E protein transcription factors and their inhibitors, Id proteins, are important regulators of both CD4+ and CD8+ T cell responses; however, their regulation at the protein level has not been explored. Recently, the deubiquitinase USP1 was shown to stabilize Id2 and modulate cellular differentiation in osteosarcomas. Here, we investigated a role for Usp1 in posttranslational control of Id2 and Id3 in T cells. We show that Usp1 was upregulated in T cells following activation in vitro or following infection in vivo, and the extent of Usp1 expression correlated with the degree of T cell expansion. Usp1 directly interacted with Id2 and Id3 following T cell activation. However, Usp1-deficiency did not impact Id protein abundance in effector T cells or alter effector CD8+ T cell expansion or differentiation following a primary infection. Usp1 deficiency did result in a gradual loss of memory cells over time and impaired accumulation and altered differentiation following a secondary infection. Together, these results identify Usp1 as a player in modulating recall responses at the protein level and highlight differences in regulation of T cell responses between primary and subsequent infection encounters. Finally, our observations reveal that differential regulation of Id2/3 proteins between immune vs non-immune cell types.

Background and aims Neointimal hyperplasia resulting from pathological vascular smooth muscle cells (VSMCs) activation is a common pathophysiological basis for numerous proliferative vascular diseases, such as restenosis. Suv39h1, an important transcription suppressor, may be involved in this process. Herein, we investigated the role of Suv39h1 in pathological intimal hyperplasia and its possible mechanisms in vitro and in vivo . Methods An adenovirus vector for Suv39h1 overexpression and a lentiviral vector for its downregulation were constructed and used to transfect cultured VSMCs in vitro . The functional changes in VSMCs stimulated by angiotensin II (Ang II) were observed and the possible mechanism was investigated. Additionally, rat carotid arteries with balloon injury were locally transfected with these viral vectors and changes in neointima formation, proliferating cell nuclear antigen (Pcna) expression and collagen deposition were examined. Results Upon Ang II stimulation, the expression of Suv39h1 and inhibitor of DNA binding 3 (Id3) was significantly increased. Suv39h1 downregulation inhibited Ang II-stimulated migration and proliferation of VSMCs, antagonized the production of Id3 and promoted p21 and p27Kip1 expression. In contrast, Suv39h1 overexpression had the opposite effects. Suv39h1 regulated the transcription of p21 and p27Kip1 by controlling H3K9me3 in the proximal promoter regions. Consistent with the VSMCs results, Suv39h1 and Id3 expression was significantly increased in blood vessels after balloon injury. Suv39h1 downregulation inhibited intimal hyperplasia, and attenuated Pcna expression and collagen synthesis in the intima, while Suv39h1 overexpression had the opposite effects. Conclusions Suv39h1 downregulation effectively inhibited neointimal hyperplasia after vascular injury.

Significance PD-1+ TCF1+ stem-like CD8 T cells are critical for maintaining the T cell response during chronic viral infection and cancer, and provide the proliferative burst seen after PD-1 immunotherapy. These cells undergo a slow self-renewal and also give rise to the more terminally differentiated and exhausted CD8 T cells. Here we define the epigenetic landscape of the stem-like CD8 T cells and their more differentiated progeny. These 2 CD8 T cell subsets from chronically infected mice showed substantial differences, but also shared common features that were distinct from the epigenetic signature of effector and memory CD8 T cells generated after an acute viral infection. This information will be useful in targeting epigenetic changes to improve current immunotherapies., We have recently defined a novel population of PD-1 (programmed cell death 1)+ TCF1 (T cell factor 1)+ virus-specific CD8 T cells that function as resource cells during chronic LCMV infection and provide the proliferative burst seen after PD-1 blockade. Such CD8 T cells have been found in other chronic infections and also in cancer in mice and humans. These CD8 T cells exhibit stem-like properties undergoing self-renewal and also differentiating into the terminally exhausted CD8 T cells. Here we compared the epigenetic signature of stem-like CD8 T cells with exhausted CD8 T cells. ATAC-seq analysis showed that stem-like CD8 T cells had a unique signature implicating activity of HMG (TCF) and RHD (NF-κB) transcription factor family members in contrast to higher accessibility to ETS and RUNX motifs in exhausted CD8 T cells. In addition, regulatory regions of the transcription factors Tcf7 and Id3 were more accessible in stem-like cells whereas Prdm1 and Id2 were more accessible in exhausted CD8 T cells. We also compared the epigenetic signatures of the 2 CD8 T cell subsets from chronically infected mice with effector and memory CD8 T cells generated after an acute LCMV infection. Both CD8 T cell subsets generated during chronic infection were strikingly different from CD8 T cell subsets from acute infection. Interestingly, the stem-like CD8 T cell subset from chronic infection, despite sharing key functional properties with memory CD8 T cells, had a very distinct epigenetic program. These results show that the chronic stem-like CD8 T cell program represents a specific adaptation of the T cell response to persistent antigenic stimulation.

Development of mammalian ovarian follicles is promoted by the combined action of endocrine cues and paracrine factors. Follicle stimulating hormone (FSH), through the action of cAMP drives follicular growth and development. The oocyte secretes powerful growth factors such as bone morphogenetic protein 15 (BMP15) to regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation through the activation of SMAD1/5/8. This study investigated the role of the cAMP signalling pathway on SMAD1/5/8 action in human granulosa cells. Cyclic AMP enhanced BMP15-induction of a SMAD1/5/8-specific BRE reporter. Moreover, in the absence of BMP ligand, cAMP also activated SMAD1/5/8-induced BRE activity. Cyclic AMP increased canonical downstream targets of BMP signalling such as inhibitor of differentiation (ID) mRNA expression. The observed effects were not mediated by secretion of BMPs as cAMP did not promote BMP ligand mRNA expression and a BMP extracellular antagonist, the BMP type II receptor ectodomain, did not affect cAMP-induced ID mRNA expression. Finally, the ERK1/2 pathway was shown to be required for the maintenance of cAMP-induced SMAD1/5/8 activity. Together our results suggest a novel and non-canonical pathway for cAMP signalling in human granulosa cells. Cyclic AMP appears to promote SMAD1/5/8 pathway activity intracellularly and has the ability to activate canonical SMAD1/5/8 downstream targets. Our results add another layer of complexity to the interactions between endocrine signalling and oocyte-secreted BMP ligands during folliculogenesis. Given the importance of both cAMP and SMAD1/5/8 pathways in follicular development, these interactions are likely required for the fine-tuning of oocyte paracrine signalling by endocrine stimuli.

Osteoblasts are versatile cells involved in multiple whole-body processes, including bone formation and immune response. Secretory amounts and patterns of osteoblast-derived proteins such as osteopontin (OPN) and osteocalcin (OCN) modulate osteoblast function. However, the regulatory mechanism of OPN and OCN expression remains unknown. Here, we demonstrate that p54/p46 c-jun N-terminal kinase (JNK) inhibition suppresses matrix mineralization and OCN expression but increases OPN expression in MC3T3-E1 cells and primary osteoblasts treated with differentiation inducers, including ascorbic acid, bone morphogenic protein-2, or fibroblast growth factor 2. Preinhibition of JNK before the onset of differentiation increased the number of osteoblasts that highly express OPN but not OCN (OPN-OBs), indicating that JNK affects OPN secretory phenotype at the early stage of osteogenic differentiation. Additionally, we identified JNK2 isoform as being critically involved in OPN-OB differentiation. Microarray analysis revealed that OPN-OBs express characteristic transcription factors, cell surface markers, and cytokines, including glycoprotein hormone α2 and endothelial cell-specific molecule 1. Moreover, we found that inhibitor of DNA binding 4 is an important regulator of OPN-OB differentiation and that dual-specificity phosphatase 16, a JNK-specific phosphatase, functions as an endogenous regulator of OPN-OB induction. OPN-OB phenotype was also observed following LPS from

Antigen-specific Th1 cells could be a passage to the infection sites during infection to execute effector functions, such as help CD8+ T cells to localize in these sites by secretion of anti-viral cytokines-IFN-γ or direct cytotoxicity of antigen-bearing cells. However, the molecular components that modulate Th1 cell differentiation and function in response to viral infection remain incompletely understood. Here, we reported that both inhibitor of DNA binding 3(Id3) protein and inhibitor of DNA binding 2(Id2) protein promoted Th1 cell differentiation. Depletion of Id3 or Id2 led to severe defect of Th1 cell differentiation during influenza virus infection. Whereas depletion of both Id3 and Id2 in CD4+ T cells restrained Th1 cell differentiation to a greater extent, indicating that Id3 and Id2 nonredundantly regulate Th1 cell differentiation. Moreover, deletion of E-proteins, the antagonists of Id proteins, greatly enhanced Th1 cell differentiation. Mechanistic study indicated that E-proteins suppressed Th1 cell differentiation by directly binding to the regulatory elements of Th1 cell master regulator T-bet and regulate T-bet expression. Thus, our findings identified Id-protein's importance for Th1 cells and clarified the nonredundant role of Id3 and Id2 in regulating Th1 cell differentiation, providing novel insight that Id3-Id2-E protein axis are essential for Th1 cell polarization.

Foxp3+ regulatory T (TR) cells are phenotypically and functionally diverse and broadly distributed in lymphoid and nonlymphoid tissues. However, the pathways guiding the differentiation of tissue-resident TR cell populations have not been well defined. By regulating E-protein function, Id3 controls the differentiation of CD8+ effector T cells and is essential for TR cell maintenance and function. We show that dynamic expression of Id3 helps define three distinct mouse TR cell populations: Id3+CD62LhiCD44lo central TR cells, Id3+CD62LloCD44hi effector TR (eTR) cells, and Id3− eTR cells. Adoptive transfer experiments and transcriptome analyses support a stepwise model of differentiation from Id3+ central TR to Id3+ eTR to Id3− eTR cells. Furthermore, Id3− eTR cells have high expression of functional inhibitory markers and a transcriptional signature of tissue-resident TR cells. Accordingly, Id3− eTR cells are highly enriched in nonlymphoid organs but virtually absent from blood and lymph. Thus, we propose that tissue-resident TR cells develop in a multistep process associated with Id3 downregulation.