Id1 expression in kidney endothelial cells protects against diabetes-induced microvascular injury.

The inhibitor of differentiation (Id) transcription regulators, which are induced in response to oxidative stress, promote cell proliferation and inhibit senescence. Inhibitor of differentiation 1 (Id1) expression is limited to endothelial cells (EC) in the normal mouse kidney and is required for a normal response to injury. Endothelial dysfunction leads to the development of diabetic nephropathy, and so, we hypothesized that endothelial Id1 may help protect against hyperglycemia-induced microvascular injury and nephropathy. Here, we tested this hypothesis by using streptozotocin to induce diabetes in Id1 knockout (KO) mice and WT B6;129 littermates and examining the mice at 3 months. Expression of Id1 was observed to be increased 15-fold in WT kidney EC, and Id1 KO mice exhibited increased mesangial and myofibroblast proliferation, matrix deposition, and albuminuria compared with WT mice. Electron microscopy demonstrated peritubular capillary EC injury and lumen narrowing, and fluorescence microangiography showed a 45% reduction in capillary perfusion area with no reduction in CD31-stained areas in Id1 KO mice. Microarray analysis of EC isolated from WT and KO control and diabetic mice demonstrated activation of senescence pathways in KO cells. Kidneys from KO diabetic mice showed increased histological expression of senescence markers. In addition, premature senescence in cultured KO EC was also seen in response to oxidative stress. In conclusion, endothelial Id1 upregulation with hyperglycemia protects against microvascular injury and senescence and subsequent nephropathy.

Study of As2O3 regulating proliferation and apoptosis of Tca8113 cells by inhibiting the expression of Id-1.

OBJECTIVE: Our study aims to investigate the effect of arsenic trioxide (As2O3) on proliferation and apoptosis of Tca8113 tongue squamous carcinoma cells. METHODS: Cell proliferation and the expression of Id-1 mRNA in Tca8113 cells after treatment with different concentrations of As2O3 were detected by MTT and qRT-PCR, respectively. The expression of Id-1, cell proliferation and apoptosis in Id-1 silencing Tca8113 cells were detected by qRT-PCR, Western blot, MTT and flow cytometry, respectively. The pcDNA 3.1-Id-1 overexpression vector was transfected into Tca8113 cells combination with 3 µmol/L As2O3. The detection of cell proliferation, apoptosis and Caspase-3, Bax and Bcl-2 protein expression in transfected Tca8113 cells were performed by MTT, flow cytometry and Western blot assay, respectively. RESULTS: As2O3 of different concentration could inhibit the proliferation of Tca8113 cells and IC50 value was 3.004 ± 0.2379 µmol/L. The expression of Id-1 mRNA was down-regulated in Tca8113 cells treated with 3 µmol/L As2O3 for 48 h. The results of qRT-PCR, Western blot, MTT and flow cytometry indicated that the expression level of Id-1 and cell proliferation ability were decreased while the apoptosis rate was increased in Tca8113 cells after transfection of Id-1 siRNA. Overexpression of Id-1 could attenuate the inhibition or promotion of As2O3 on proliferation, apoptosis and Caspase-3, Bax and Bcl-2 protein expression in Tca8113 cells. CONCLUSION: As2O3 could regulate the proliferation and apoptosis of Tca8113 cells by inhibiting the expression of Id-1.

Knockdown of inhibitor of differentiation 1 suppresses proliferation and induces apoptosis by inactivating PI3K/Akt/mTOR signaling in hemangioma-derived endothelial cells.

Hemangioma (HA) is one of the commonest benign vascular neoplasms of infancy. Inhibitor of differentiation 1 (ID-1) has been reported to be an oncogene in multiple cancers. However, the role of ID-1 and its molecular mechanism in HA progression have not been elucidated. In the present study, we found that ID-1 expression at mRNA and protein levels was up-regulated in HA-derived endothelial cells (HDECs). Knockdown of ID-1 inhibited proliferation, facilitated apoptosis, and enhanced propranolol cytotoxicity in HDECs. Knockdown of ID-1 decreased the protein levels of phospholyrated protein kinase-B (Akt) and phospholyrated mammalian target of rapamycin (mTOR). Inhibition of PI3K/Akt/mTOR pathway by LY294002 abrogated ID-1-mediated pro-proliferation and anti-apoptosis effects in HDECs. In conclusion, knockdown of ID-1 suppressed proliferation and promoted apoptosis by inactivating phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling in HDECs, shedding light on the function of ID-1 in HA progression and highlighting the therapeutic value of ID-1 for HA.

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

Defining mechanisms that maintain tissue stem cells during homeostasis, stress, and aging is important for improving tissue regeneration and repair and enhancing cancer therapies. Here, we show that Id1 is induced in hematopoietic stem cells (HSCs) by cytokines that promote HSC proliferation and differentiation, suggesting that it functions in stress hematopoiesis. Genetic ablation of Id1 increases HSC self-renewal in serial bone marrow transplantation (BMT) assays, correlating with decreases in HSC proliferation, mitochondrial biogenesis, and reactive oxygen species (ROS) production. Id1-/- HSCs have a quiescent molecular signature and harbor less DNA damage than control HSCs. Cytokines produced in the hematopoietic microenvironment after γ-irradiation induce Id1 expression. Id1-/- HSCs display a blunted proliferative response to such cytokines and other inducers of chronic proliferation including genotoxic and inflammatory stress and aging, protecting them from chronic stress and exhaustion. Thus, targeting Id1 may be therapeutically useful for improving HSC survival and function during BMT, chronic stress, and aging.

Combined Id1 and Id3 Deletion Leads to Severe Erythropoietic Disturbances.

The Inhibitor of DNA Binding (Id) proteins play a crucial role in regulating hematopoiesis and are known to interact with E proteins and the bHLH family of transcription factors. Current efforts seek to elucidate the individual roles of Id members in regulating hematopoietic development and specification. However, the nature of their functional redundancies remains elusive since ablation of multiple Id genes is embryonically lethal. We developed a model to test this compensation in the adult. We report that global Id3 ablation with Tie2Cre-mediated conditional ablation of Id1 in both hematopoietic and endothelial cells (Id cDKO) extends viability to 1 year but leads to multi-lineage hematopoietic defects including the emergence of anemia associated with defective erythroid development, a novel phenotype unreported in prior single Id knockout studies. We observe decreased cell counts in the bone marrow and splenomegaly to dimensions beyond what is seen in single Id knockout models. Transcriptional dysregulation of hematopoietic regulators observed in bone marrow cells is also magnified in the spleen. E47 protein levels were elevated in Id cDKO bone marrow cell isolates, but decreased in the erythroid lineage. Chromatin immunoprecipitation (ChIP) studies reveal increased occupancy of E47 and GATA1 at the promoter regions of ß-globin and E2A. Bone marrow transplantation studies highlight the importance of intrinsic Id signals in maintaining hematopoietic homeostasis while revealing a strong extrinsic influence in the development of anemia. Together, these findings demonstrate that loss of Id compensation leads to dysregulation of the hematopoietic transcriptional network and multiple defects in erythropoietic development in adult mice.

Regulation of AKT signaling by Id1 controls t(8;21) leukemia initiation and progression.

Transcriptional regulators are recurrently altered through translocations, deletions, or aberrant expression in acute myeloid leukemia (AML). Although critically important in leukemogenesis, the underlying pathogenetic mechanisms they trigger remain largely unknown. Here, we identified that Id1 (inhibitor of DNA binding 1) plays a pivotal role in acute myeloid leukemogenesis. Using genetically modified mice, we found that loss of Id1 inhibited t(8;21) leukemia initiation and progression in vivo by abrogating protein kinase B (AKT)1 activation, and that Id1 interacted with AKT1 through its C terminus. An Id1 inhibitor impaired the in vitro growth of AML cells and, when combined with an AKT inhibitor, triggered even greater apoptosis and growth inhibition, whereas normal hematopoietic stem/progenitor cells were largely spared. We then performed in vivo experiments and found that the Id1 inhibitor significantly prolonged the survival of t(8;21)(+) leukemic mice, whereas overexpression of activated AKT1 promoted leukemogenesis. Thus, our results establish Id1/Akt1 signaling as a potential therapeutic target in t(8;21) leukemia.

Inhibitor of differentiation proteins protect against oxidative stress by regulating the antioxidant-mitochondrial response in mouse beta cells.

AIMS/HYPOTHESIS: Oxidative stress is implicated in beta cell glucotoxicity in type 2 diabetes. Inhibitor of differentiation (ID) proteins are transcriptional regulators induced by hyperglycaemia in islets, but the mechanisms involved and their role in beta cells are not clear. Here we investigated whether or not oxidative stress regulates ID levels in beta cells and the role of ID proteins in beta cells during oxidative stress. METHODS: MIN6 cells were cultured in H2O2 or ribose to induce oxidative stress. ID1, ID3 and small MAF proteins (MAFF, MAFG and MAFK) were inhibited using small interfering RNA. Isolated islets from Id1(-/-), Id3(-/-) and diabetic db/db mice were used. RESULTS: ID1-4 expression was upregulated in vivo in the islets of diabetic db/db mice and stimulated in vitro by ribose and H2O2. Id1/3 inhibition reduced the expression of multiple antioxidant genes and potentiated oxidative stress-induced apoptosis. This finding was associated with increased levels of intracellular reactive oxygen species, altered mitochondrial morphology and reduced expression of Tfam, which encodes a mitochondrial transcription factor, and respiratory chain components. Id1/3 inhibition also reduced the expression of small MAF transcription factors (MafF, MafG and MafK), interacting partners of nuclear factor, erythroid 2-like 2 (NFE2L2), master regulator of the antioxidant response. Inhibition of small MAFs reduced the expression of antioxidant genes and potentiated oxidative stress-induced apoptosis, thus recapitulating the effects of Id1/3 inhibition. CONCLUSIONS/INTERPRETATION: Our study identifies IDs as a novel family of oxidative stress-responsive proteins in beta cells. IDs are crucial regulators of the adaptive antioxidant-mitochondrial response that promotes beta cell survival during oxidative stress through a novel link to the NFE2L2-small MAF pathway.

Chronic TLR signaling impairs the long-term repopulating potential of hematopoietic stem cells of wild type but not Id1 deficient mice.

Hematopoietic stem cells (HSCs) maintain life-long blood supply but are inevitably exposed to various inflammatory stimuli, which have been shown to be harmful for HSC integrity but the mediators of the deleterious effects have not been fully identified. Here, we show that daily injection of mice with 1 µg of LPS for 30 days triggers a storm of inflammatory cytokines. LPS injection also stimulated the transcription of the Id1 gene in HSCs in vivo but not in vitro, suggesting an indirect effect. To determine the effects of LPS treatment on HSC function and to evaluate the significance of Id1 expression, we assess the repopulating potential of wild type and Id1 deficient mice, which were subjected to a 30 day regimen of LPS treatment. We found that LPS caused dramatic reduction in the long-term but not short-term repopulating activity of wild type but not Id1 deficient HSC. This treatment also led to increases in HSC counts, decreases in BrdU-label retention and disturbance of quiescence detected by Ki67 staining in wild type but not Id1 deficient mice. Together, it appears that Id1, at least in part, plays a role in LPS-induced damage of HSC integrity.

Tubular cell dedifferentiation and peritubular inflammation are coupled by the transcription regulator Id1 in renal fibrogenesis.

During renal fibrogenesis, tubular epithelial-mesenchymal transition is closely associated with peritubular inflammation; however, it is not clear whether these two processes are connected. We previously identified the inhibitor of differentiation-1 (Id1), a dominant negative antagonist of basic helix-loop-helix transcription factors, as a major trigger of tubular cell dedifferentiation after injury. Id1 was induced selectively in degenerated proximal tubule and collecting duct epithelia after injury and was present in both the cytoplasm and nucleus, suggesting shuttling between these two compartments. Interestingly, the upregulation of Id1 was associated with peritubular inflammation in mouse and human nephropathies. In vitro, Id1 potentiated NF-κB signaling and augmented RANTES expression in kidney epithelial cells, which led to an enhanced recruitment of inflammatory cells. Id1 also induced Snail1 expression and triggered tubular epithelial dedifferentiation. In vivo, genetic ablation of Id1 in mice reduced peritubular inflammation and decreased tubular expression of RANTES following ureteral obstruction. Mice lacking Id1 were also protected against myofibroblast activation and matrix expression, leading to a reduced total collagen deposition in obstructive nephropathy. Thus, these results indicate that Id1 shuttles between nucleus and cytoplasm, and promotes peritubular inflammation and tubular epithelial dedifferentiation, suggesting that these two events are intrinsically coupled during renal fibrogenesis.

Developmental ablation of Id1 and Id3 genes in the vasculature leads to postnatal cardiac phenotypes.

The Id1 and Id3 genes play major roles during cardiac development, despite their expression being confined to non-myocardial layers (endocardium-endothelium-epicardium). We previously described that Id1Id3 double knockout (dKO) mouse embryos die at mid-gestation from multiple cardiac defects, but early lethality precluded the studies of the roles of Id in the postnatal heart. To elucidate postnatal roles of Id genes, we ablated the Id3 gene and conditionally ablated the Id1 gene in the endothelium to generate conditional KO (cKO) embryos. We observed cardiac phenotypes at birth and at 6 months of age. Half of the Id cKO mice died at birth. Postnatal demise was associated with cardiac enlargement and defects in the ventricular septum, trabeculation and vasculature. Surviving Id cKO mice exhibited fibrotic vasculature, cardiac enlargement and decreased cardiac function. An abnormal vascular response was also observed in the healing of excisional skin wounds of Id cKO mice. Expression patterns of vascular, fibrotic and hypertrophic markers were altered in the Id cKO hearts, but addition of Insulin-Like Growth Factor binding protein-3 (IGFbp3) reversed gene expression profiles of vascular and fibrotic, but not hypertrophic markers. Thus, ablation of Id genes in the vasculature leads to distinct postnatal cardiac phenotypes. These findings provide important insights into the role/s of the endocardial network of the endothelial lineage in the development of cardiac disease, and highlight IGFbp3 as a potential link between Id and its vascular effectors.

Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration.

During embryogenesis, endothelial cells induce organogenesis before the development of circulation. These findings suggest that endothelial cells not only form passive conduits to deliver nutrients and oxygen, but also establish an instructive vascular niche, which through elaboration of paracrine trophogens stimulates organ regeneration, in a manner similar to endothelial-cell-derived angiocrine factors that support haematopoiesis. However, the precise mechanism by which tissue-specific subsets of endothelial cells promote organogenesis in adults is unknown. Here we demonstrate that liver sinusoidal endothelial cells (LSECs) constitute a unique population of phenotypically and functionally defined VEGFR3(+)CD34(-)VEGFR2(+)VE-cadherin(+)FactorVIII(+)CD45(-) endothelial cells, which through the release of angiocrine trophogens initiate and sustain liver regeneration induced by 70% partial hepatectomy. After partial hepatectomy, residual liver vasculature remains intact without experiencing hypoxia or structural damage, which allows study of physiological liver regeneration. Using this model, we show that inducible genetic ablation of vascular endothelial growth factor (VEGF)-A receptor-2 (VEGFR2) in the LSECs impairs the initial burst of hepatocyte proliferation (days 1-3 after partial hepatectomy) and subsequent reconstitution of the hepatovascular mass (days 4-8 after partial hepatectomy) by inhibiting upregulation of the endothelial-cell-specific transcription factor Id1. Accordingly, Id1-deficient mice also manifest defects throughout liver regeneration, owing to diminished expression of LSEC-derived angiocrine factors, including hepatocyte growth factor (HGF) and Wnt2. Notably, in in vitro co-cultures, VEGFR2-Id1 activation in LSECs stimulates hepatocyte proliferation. Indeed, intrasplenic transplantation of Id1(+/+) or Id1(-/-) LSECs transduced with Wnt2 and HGF (Id1(-/-)Wnt2(+)HGF(+) LSECs) re-establishes an inductive vascular niche in the liver sinusoids of the Id1(-/-) mice, initiating and restoring hepatovascular regeneration. Therefore, in the early phases of physiological liver regeneration, VEGFR2-Id1-mediated inductive angiogenesis in LSECs through release of angiocrine factors Wnt2 and HGF provokes hepatic proliferation. Subsequently, VEGFR2-Id1-dependent proliferative angiogenesis reconstitutes liver mass. Therapeutic co-transplantation of inductive VEGFR2(+)Id1(+)Wnt2(+)HGF(+) LSECs with hepatocytes provides an effective strategy to achieve durable liver regeneration.

Id1 overexpression induces tetraploidization and multiple abnormal mitotic phenotypes by modulating aurora A.

The basic helix-loop-helix transcription factor, Id1, was shown to induce tetraploidy in telomerase-immortalized nasopharyngeal epithelial cells in this study. Using both transient and stable Id1-expressing cell models, multiple mitotic aberrations were detected, including centrosome amplification, binucleation, spindle defects, and microtubule perturbation. Many of these abnormal phenotypes have previously been reported in cells overexpressing Aurora A. Further experiments showed that Id1 could stabilize Aurora A, whereas knocking down Aurora A expression in Id1-expressing cells could rescue some of the mitotic defects. The mechanisms by which Aurora A could be modulated by Id1 were explored. DNA amplification of the Aurora A locus was not involved. Id1 could only weakly activate the transcriptional activity of the Aurora A promoter. We found that Id1 overexpression could affect Aurora A degradation, leading to its stabilization. Aurora A is normally degraded from mitosis exit by the APC/C(Cdh1)-mediated proteasomal proteolysis pathway. Our results revealed that Id1 and Cdh1 are binding partners. The association of Id1 and Cdh1 was found to be dependent on the canonical destruction box motif of Id1, the increased binding of which may compete with the interaction between Cdh1 and Aurora A, leading to stabilization of Aurora A in Id1-overexpressing cells.

Id sustains Hes1 expression to inhibit precocious neurogenesis by releasing negative autoregulation of Hes1.

Negative bHLH transcription factor Hes1 can inhibit neural stem cells (NSCs) from precocious neurogenesis through repressing proneural gene expression; therefore, sustenance of Hes1 expression is crucial for NSC pool maintenance. Here we find that Ids, the dominant-negative regulators of proneural proteins, are expressed prior to proneural genes and share an overlapping expression pattern with Hes1 in the early neural tube of chick embryos. Overexpression of Id2 in the chick hindbrain upregulates Hes1 expression and inhibits proneural gene expression and neuronal differentiation. By contrast, Hes1 expression decreases, proneural gene expression expands, and neurogenesis occurs precociously in Id1;Id3 double knockout mice and in Id1-3 RNAi-electroporated chick embryos. Mechanistic studies show that Id proteins interact directly with Hes1 and release the negative feedback autoregulation of Hes1 without interfering with its ability to affect other target genes. These results indicate that Id proteins participate in NSC maintenance through sustaining Hes1 expression in early embryos.

Id1, but not Id3, directs long-term repopulating hematopoietic stem-cell maintenance.

E-proteins are widely expressed basic helix-loop-helix (HLH) transcription factors that regulate differentiation in many cell lineages, including lymphoid, muscle, and neuronal cells. E-protein function is controlled by HLH inhibitors such as Id and SCL/TAL1 proteins, which recently have been suggested to play a role in hematopoietic stem cell (HSC) differentiation. However, the precise stages when these proteins are expressed and their specific functions are not entirely clear. Using a knock-in mouse model where the sequence for the enhanced green fluorescent protein (GFP) was inserted downstream of the Id1 promoter, we were able to track Id1 expression on an individual cell basis and detected Id1 expression in long-term repopulating HSCs (LT-HSCs). Functional assays showed that the Id1/GFP(+)Lin(-)Sca1(+)c-kit(Hi) population was highly enriched for LT-HSCs. Consistent with this expression pattern, Id1 deficiency led to a 2-fold reduction in the number of LT-HSCs defined as Lin(-)Sca1(+)c-kit(Hi)CD48(-)CD150(+). Primary bone marrow transplantation studies revealed that Id1 is dispensable for short-term engraftment. In contrast, both Id1(-/-) whole bone marrow and Lin(-)Sca1(+)c-kit(Hi)Thy1.1(Lo)-enriched HSCs, but not Id3(-/-) marrow, displayed impaired engraftment relative to wild-type controls in secondary transplantation assays. These findings suggest a unique role for Id1 in LT-HSC maintenance and hematopoietic development.