The Experts below are selected from a list of 589203 Experts worldwide ranked by ideXlab platform
Robert Benezra - One of the best experts on this subject based on the ideXlab platform.
-
ID1, Id2, Id3, and Id4 in adult mouse brain lateral wall V-SVZ neural stem cells
2020Co-Authors: Hyung-song Nam, Mario Capecchi, Robert BenezraAbstract:An R script to analyze the Mizrak et al., 2019 dataset.Clusters. Selected marker genes RNA in clusters.Markers for selected subclusters identified by Seurat.To validate the AverageExpression command results, RT-qPCR measurements from Morizur et al., 2018 (https://doi.org/10.1016/j.stemcr.2018.06.005) were utilized. The trends were very similar, suggesting that normalized scRNA-seq datasets can also reveal the average RNA expression level of cell populations otherwise obtained by FACS sorting and RT-qPCR.The relative RNA abundance in the lateral wall V-SVZ qNSC's of ID1, Id2, Id3, and Id4 seems to be Id2 > Id3 > Id4 > ID1. How the RNA levels correlate to protein levels is unclear, although the ID1 protein in particular was measured directly by knock-in C-terminal fusion of the Venus yellow fluorescent protein (https://doi.org/10.1016/j.stem.2009.08.017, https://doi.org/10.1016/j.ccr.2011.11.025, https://doi.org/10.1242/jcs.096198, https://doi.org/10.1016/j.stemcr.2014.09.012, https://doi.org/10.7554/elife.01197, https://doi.org/10.1016/j.devcel.2019.05.032). ==============The material from my PhD thesis work I put up here after going through them again after more than ten years are (1) copied and pasted from my PhD thesis, (2) data from my PhD thesis work (Immunofluorescence images, Southern blots, PCR gels, restriction digests, and flow data), and (3) re-analysis of the data using modern software (flow data and sequence analyses for the knock-in allele targeting vectors and Southern blots).Mirax epifluorescence scans of coronal sections of >6 week old wildtype mouse brain. ID1 (green). Ki-67 or Mcm2 (red). DAPI (blue). ID1 and Ki-67 or Mcm2 merged.A confocal z-stack from coronal section of a wildtype mouse brain V-SVZ, >6 week old. A maximum projection image montage and a movie of the z-stack. ID1 (green). Mcm2 (red). DAPI (blue).A confocal z-stack from coronal section of a wildtype mouse brain V-SVZ, >6 week old. Perfused 48 h after the cessation of Ara-C infusion for 6 days, as described in Nam and Benezra, 2009. EdU was injected 30 min before the perfusion. A maximum projection image montage and a movie of the z-stack. ID1 (green). EdU (red). DAPI (blue). The small dots are probably non-specific protein precipitates from the block buffer.A confocal z-stack from coronal section of a wildtype mouse brain V-SVZ, >6 week old. A maximum projection image montage and movies of the z-stack. ID1 (green). S100beta (red). DAPI (blue).Some self-explanatory images.Production of the ID1-floxed allele mouse (http://www.informatics.jax.org/allele/MGI:4366910), the ID1-Venus allele mouse (http://www.informatics.jax.org/allele/MGI:4366905), the ID1-IRES-creERT2 allele mouse (http://www.informatics.jax.org/allele/MGI:4366863), and the ROSA26-StLa allele mouse (http://www.informatics.jax.org/allele/MGI:4366911).There were two variants of the ID1 targeting vector. The first variant utilized a long 5' arm and a DTA cassette. The second variant utilized a shorter 5' arm and DTA and TK cassettes. The lengths of the homology arms in the first variant were from Yan et al., 1997 (https://doi.org/10.1128/mcb.17.12.7317). The actual genomic DNA was subcloned from RPCI-23 C57BL/6J mouse genome BAC library using recombineering.I would put up the Sanger sequencing files of the targeting vectors if I could, but I'm not sure if I have the intellectual property rights to do that. Regardless, I have re-analyzed the sequencing results of the ID1 targeting vectors, and did not find any errors. I did not go through the ROSA26-StLa targeting vector sequencing again. By the way, these targeting vectors were constructed on a high copy backbone. With the large genomic insert, the plasmid DNA of the final construct was very difficult to prepare because the E. coli harboring it grew very slowly, and the plasmid was prone to rearrangements by recombination. This sometimes happens - see https://www.addgene.org/61580/ for comments from the Hongkui Zeng laboratory. To make it work, plasmid prep from every culture of the clone had to be checked for recombination by restriction digests before using. Preps of recombined plasmids had to be discarded. The low copy backbone plasmids are much easier to work with because they don't behave this way. For anybody still doing gene targeting, I would recommend them (see https://doi.org/10.6084/m9.figshare.c.5277341.v12 for links to Addgene).Flow cytometry data. The figure pdf shows the gates that were utilized to detect ID1-Venus+ cells from dissociated V-SVZ cells as well as ID1-Venus+ cells in cultured V-SVZ cells. The zip files are the actual FCS files. The file names were modified to be descriptive.Tried a wider gate for ID1-Venus and Gfap detection. Could see more in the double-positive gate, but the larger cells might be more autofluorescent.As in the paper, there were neural ID1-Venus+ cells as well as endothelial ID1-Venus+ cells in the V-SVZ (mice >6 weeks of age). Culturing the V-SVZ cells in neurosphere-forming media with EGF and FGF-2 was a quick way to enrich for the neural lineage cells without FACS, etc. In the cultures of neural lineage cells of the V-SVZ thus obtained, there were ID1-Venus+ and ID1-Venus- cells that could be discerned. The ID1-Venus-high cells formed self-renewing neurospheres, etc as described in the paper. As an aside, the ID1-Venus fusion protein reporter actually generated a very sensitive read-out of the ID1 protein. These are why I think so. The ID1 protein was very difficult to detect in the brain tissue (i.e., no signal with conventional indirect immunofluorescence in both cell types). I could only visualize it after Tyramide Signal Amplification. The labeling efficiency with the ID1IRES-creERT2 allele was also somewhat low. Although other reasons are also possible, these suggested to me low protein expression level. Then, there may not be so many copies of the ID1-Venus protein in the cell. Yet, it was detectable with flow cytometers. If the Discussion of the Nam and Benezra, 2009 paper wasn't clear, I note here that even though the absolute level of the ID1 protein may be low, its level is highest in the stem/progenitor cells if one considers the relative levels along the neurogenic lineage.ID1-Venus fluorescence from adherent NSPC cultures.Two confocal images of YFP+ cells from ventricular wall whole mount of ID1IRES-creERT2/+; ROSA26LSL-YFP/+ mice that were scored to be "B1" cells from Mirzadeh et al., 2008 (https://doi.org/10.1016/j.stem.2008.07.004). Previously, the whole mount immunfluorescence technique wasn't as optimized as it is now, so the images weren't so clear. However, putting the previous work in context with my more recent work, what is clear is that the cells labeled by the ID1IRES-creERT2 allele were different from the cells labeled by the Lrig1T2A-iCreERT2 allele (see https://doi.org/10.6084/m9.figshare.12731900.v8). So how is it possible to get Lrig1 out of ID1high cells when they label cells with different morphologies? Although it wasn't apparent with the previous technique, more recent results suggest that ID1 also labels the cells labeled by Lrig1 (unpublished preliminary observation). So, a working model is that there are at least two different types of stem cells: ID1 reveals both, whereas Lrig1 reveals mostly one subset. ==============ID1+ cells outside the V-SVZ.Two coronal section images from hippocampus of ID1IRES-creERT2/+; ROSA26StLa/+ mouse. 1 month after tamoxifen inductions. Non-endothelial X-gal+ cells (from tauLacZ) in the dentate gyrus.A confocal image from coronal section of hippocampus of ID1IRES-creERT2/+; ROSA26StLa/+ mouse. 1 month after tamoxifen inductions. Tau-b-gal (green), NeuN (red), and DAPI (blue).A confocal image from a coronal section of wildtype mouse embryo (~E12.5) spinal cord. ID1 (green), Nestin (red), and DAPI (blue).
-
id proteins contribute to tumor development and metastatic colonization in a model of bladder carcinogenesis
Bladder cancer (Amsterdam Netherlands), 2015Co-Authors: Marta Garciacao, Hikmat Alahmadie, Yvette Chin, Bernard H Bochner, Robert BenezraAbstract:Background: Bladder cancer is one of the most common malignant genitourinary diseases worldwide. Despite advances in surgical technique, medical oncology and radiation therapy, cure of invasive tumors remains elusive for patients with late stage disease. Therefore, new therapeutic strategies are needed to improve the response rates with regard to recurrence, invasion and metastasis. Objective: Inhibitor of DNA binding (Id) proteins have been proposed as therapeutic targets due to the key regulatory role they exert in multiple steps of cancer. We aimed to explore the role of Id proteins in bladder cancer development and the pattern of expression of Id proteins in bladder carcinomas. Methods: We used a well-established chemically induced model of bladder carcinogenesis. Wild type and Id-deficient mice were given N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in the drinking water and urinary bladder lesions were analyzed histopathologically and stained for ID1. We assessed the effects of ID1 inactivation in cultured bladder cancer cells and in a model of metastatic lung colonization. We also performed ID1 staining of human urothelial carcinoma samples and matched lymph node metastases. Results: ID1 protein was overexpressed in the BBN-induced model of bladder cancer. ID1 deficiency resulted in the development of urinary bladder tumors with areas of extensive hemorrhage and decreased invasiveness when compared to wild type mice. ID1 inactivation led to decreased cell growth in vitro and lung colonization in vivo of human bladder cancer cells. Immunohistochemistry performed on human urothelial carcinoma samples showed ID1 positive staining in both primary tumors and lymph node metastases. Conclusions: In summary, our studies reveal the physiological relevance of ID1 in bladder cancer progression and suggest that targeting ID1 may be important in the development of novel therapies for the treatment of bladder cancer.
-
expansion and maintenance of human embryonic stem cell derived endothelial cells by tgfβ inhibition is ID1 dependent
Nature Biotechnology, 2010Co-Authors: Daylon James, Robert Benezra, Daniel J Nolan, David Lyden, Marco Seandel, Tyler Janovitz, Mark J Tomishima, Lorenz Studer, N ZaninovicAbstract:Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell–specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)β at day 7 of differentiation increases hVPr-GFP+ cells by tenfold. In phase 2, TGFβ inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of ID1highVEGFR2highVE-cadherin+ ephrinB2+. Using an ID1-YFP hESC reporter line, we showed that TGFβ inhibition sustains ID1 expression in hESC-derived endothelial cells and that ID1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.
-
id genes mediate tumor reinitiation during breast cancer lung metastasis
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Gaorav P Gupta, Robert Benezra, Vivek Mittal, Jonathan Perk, Paola De Candia, Swarnali Acharyya, Katia Todorovamanova, William L Gerald, Edi Brogi, Joan MassagueAbstract:The establishment of distant metastases depends on the capacity of small numbers of cancer cells to regenerate a tumor after entering a target tissue. The mechanisms that confer this capacity remain to be defined. Here we identify a role for the transcriptional inhibitors of differentiation ID1 and Id3 as selective mediators of lung metastatic colonization in the triple negative [TN, i.e., lacking expression of estrogen receptor and progesterone receptor, and lacking Her2 (human epidermal growth factor receptor 2) amplification] subgroup of human breast cancer. Although broad expression of ID1 has recently been documented in tumors of the rare metaplastic subtype, here we report that rare ID1-expressing cells are also present in the more common TN subset of human breast tumors but not in other subtypes. We also provide evidence that ID1 expression is enriched in clinically obtained hormone receptor negative lung metastases. Functional studies demonstrate that ID1 and its closely related family member Id3 are required for tumor initiating functions, both in the context of primary tumor formation and during metastatic colonization of the lung microenvironment. In vivo characterization of lung metastatic progression reveals that ID1 and Id3 facilitate sustained proliferation during the early stages of metastatic colonization, subsequent to extravasation into the lung parenchyma. These results shed light on the proliferative mechanisms that initiate metastatic colonization, and they implicate ID1 and Id3 as mediators of this malignant function in the TN subgroup of breast cancers.
-
angiogenesis impairment in id deficient mice cooperates with an hsp90 inhibitor to completely suppress her2 neu dependent breast tumors
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Paola De Candia, Edi Brogi, David B Solit, Dilip Giri, Peter M Siegel, Adam B Olshen, William J Muller, Neal Rosen, Robert BenezraAbstract:Id proteins bind basic helix–loop–helix transcription factors and function as dominant negative inhibitors of gene expression. ID1 and Id3 are required for the recruitment of bone marrow-derived endothelial cell precursors and tumors transplanted into Id-deficient mice demonstrate impaired angiogenesis. Mouse mammary tumor virus–neu mice were bred with ID1–/–Id3+/– mice to ascertain the role of ID1 and Id3 in mammary tumorigenesis in a more physiologically relevant model. In mammary tumors from these mice, ID1 and Id3 expression was restricted to the vascular endothelium. ID1 and Id3 deficiency did not prevent or delay tumor formation but did alter tumor phenotype. The tumors that developed in the Id-deficient mice were larger and cystic with a viable rim of tumor cells surrounding a nonviable core of cellular debris. The Hsp90 chaperone protein is required for cellular survival under condition of environmental stress and for the stability of the neu oncogene. 17-Allylamino-17-demethoxygeldanamycin, an Hsp90 inhibitor, was used to treat these mice. Whereas 17-allylamino-17-demethoxygeldanamycin only modestly delayed the growth of established mammary tumors in WT mice for Id, tumor suppression was dramatically more effective in an ID1- or Id3-deficient background. These data suggest that tumorigenesis can occur in a background of defective angiogenesis but that tumors developing in such an environment may be especially sensitive to inhibitors of neu and stress-activated survival pathways. Thus angiogenesis inhibitors in combination with inhibitors of Hsp90 function should be evaluated for the treatment of advanced breast cancer.
Alan D Dandrea - One of the best experts on this subject based on the ideXlab platform.
-
small molecule inhibitors of usp1 target ID1 degradation in leukemic cells
Molecular Cancer Therapeutics, 2013Co-Authors: Helena Mistry, Grace Hsieh, Ilene Galinsky, Sara J Buhrlage, Eunmi Park, Gregory D. Cuny, Richard Stone, Nathanael S. Gray, Min Huang, Alan D DandreaAbstract:Inhibitor of DNA-binding-1 (ID1) transcription factor is essential for the proliferation and progression of many cancer types including leukemia. However, the ID1 protein has not yet been therapeutically targeted in leukemia. ID1 is normally polyubiquitinated and degraded by the proteasome. Recently, it has been shown that USP1, a ubiquitin specific protease, deubiquitinates ID1 and rescues it from proteasome degradation. Inhibition of USP1 therefore offers a new avenue to target ID1 in cancer. Here, using a Ubiquitin-Rhodamine-based high throughput screening, we identified small molecule inhibitors of USP1 and investigated their therapeutic potential for leukemia. These inhibitors blocked the deubiquitinating enzyme activity of USP1 in vitro in a dose-dependent manner with an IC50 in the high nanomolar range. USP1 inhibitors promoted the degradation of ID1 and, concurrently, inhibited the growth of leukemic cell lines in a dose dependent manner. A known USP1 inhibitor, Pimozide, also promoted ID1 degradation and inhibited growth of leukemic cells. In addition, the growth of primary Acute Myeloid Leukemia (AML) patient-derived leukemic cells was inhibited by a USP1 inhibitor. Collectively, these results indicate that the novel small molecule inhibitors of USP1 promote ID1 degradation and are cytotoxic to leukemic cells. The identification of USP1 inhibitors therefore opens up a new approach for leukemia therapy.
-
small molecule inhibitors of usp1 target ID1 degradation in leukemic cells
Molecular Cancer Therapeutics, 2013Co-Authors: Helena Mistry, Grace Hsieh, Ilene Galinsky, Sara J Buhrlage, Eunmi Park, Gregory D. Cuny, Richard Stone, Nathanael S. Gray, Min Huang, Alan D DandreaAbstract:Inhibitor of DNA binding 1 (ID1) transcription factor is essential for the proliferation and progression of many cancer types, including leukemia. However, the ID1 protein has not yet been therapeutically targeted in leukemia. ID1 is normally polyubiquitinated and degraded by the proteasome. Recently, it has been shown that USP1, a ubiquitin-specific protease, deubiquitinates ID1 and rescues it from proteasome degradation. Inhibition of USP1 therefore offers a new avenue to target ID1 in cancer. Here, using a ubiquitin-rhodamine-based high-throughput screening, we identified small-molecule inhibitors of USP1 and investigated their therapeutic potential for leukemia. These inhibitors blocked the deubiquitinating enzyme activity of USP1 in vitro in a dose-dependent manner with an IC50 in the high nanomolar range. USP1 inhibitors promoted the degradation of ID1 and, concurrently, inhibited the growth of leukemic cell lines in a dose-dependent manner. A known USP1 inhibitor, pimozide, also promoted ID1 degradation and inhibited growth of leukemic cells. In addition, the growth of primary acute myelogenous leukemia (AML) patient-derived leukemic cells was inhibited by a USP1 inhibitor. Collectively, these results indicate that the novel small-molecule inhibitors of USP1 promote ID1 degradation and are cytotoxic to leukemic cells. The identification of USP1 inhibitors therefore opens up a new approach for leukemia therapy.
-
small molecule inhibitors of usp1 target ID1 degradation in leukemic cells and cause cytotoxicity
Blood, 2013Co-Authors: Grace Hsieh, Kalindi Parmar, Ilene Galinsky, Sara J Buhrlage, Eunmi Park, Gregory D. Cuny, Richard Stone, Nathanael S. Gray, Min Huang, Alan D DandreaAbstract:ID1 (inhibitor of DNA-binding-1) is a member of the helix-loop-helix family of transcriptional regulatory proteins. The ID-family of proteins (ID1-ID4) inhibit the DNA binding of transcription factors which regulate cellular differentiation and proliferation. Accordingly, deregulation of ID proteins has been observed in many cancer types including leukemia. High levels of ID1 expression are found in primary acute myeloid leukemia (AML) samples and correlate with poor prognosis. ID1 is also identified as a common downstream target of the oncogenic tyrosine kinases, BCR-ABL, TEL-ABL and FLT3-ITD. In addition, ID1 has been shown to promote a myeloproliferative disease in mice, and knockdown of ID1 expression inhibits leukemic cell growth. Therefore, ID1 is an excellent candidate for targeted therapy in leukemia. However, suitable drugs to target ID1 have not been developed to date. ID1 is normally polyubiquitinated and degraded by the proteasome. Recently, it has been shown that USP1, a ubiquitin specific protease, deubiquitinates ID1 and rescues it from proteasome degradation. Inhibition of USP1 therefore offers a new avenue to target ID1 in cancer. Here, using a Ubiquitin-Rhodamine-based high throughput screen, we identified small molecule inhibitors of USP1 and investigated their therapeutic potential for leukemia. These inhibitors blocked the deubiquitinating enzyme activity of USP1 in vitro in a dose-dependent manner with an IC50 in the nanomolar range, and also targeted the enzyme activity of native USP1. To determine the cellular consequences of USP1 inhibition, we exposed leukemic cells to micromolar concentrations of the inhibitors and evaluated ID1 levels and survival. USP1 inhibitors promoted the degradation of ID1 and, concurrently, inhibited the growth (>90% inhibition in 24 hrs) of chronic myelogenous leukemia (CML) and AML cell lines with induction of apoptosis in a dose dependent manner. The EC50 of the inhibitors for the leukemic cell growth inhibition was approximately 1.07 μM ± 0.08 (95% Confidence Limits). Interestingly, exposure to low doses of USP1 inhibitor for 5 days in culture resulted in erythroid differentiation of K562 leukemic cells. A known USP1 inhibitor, Pimozide, also promoted ID1 degradation and inhibited growth of leukemic cells (>90% inhibition in 48 hrs), though at a higher drug concentrations as compared to the novel USP1 inhibitors. Importantly, the novel USP1 inhibitors promoted ID1 degradation and exhibited cytotoxicity (>90% death in 48 hrs) in primary AML patient-derived leukemic cells. Notably, siRNA-mediated knockdown of USP1 in K562 leukemic cells resulted in growth inhibition, increased apoptosis and cell cycle arrest. Collectively, our results demonstrate that the novel small molecule inhibitors of USP1 promote ID1 degradation and are cytotoxic to leukemic cells. The identification of USP1 inhibitors therefore opens up a new approach for leukemia therapy. Disclosures: No relevant conflicts of interest to declare.
Qingyin Zheng - One of the best experts on this subject based on the ideXlab platform.
-
hearing loss in ID1 id3 and ID1 id3 mice is associated with a high incidence of middle ear infection otitis media
Frontiers in Genetics, 2021Co-Authors: Qingyin Zheng, Tihua Zheng, Aizhen Zhang, Bin Yan, Zhaoqiang Zhang, Yan ZhangAbstract:Inhibitors of differentiation/DNA binding (Id) proteins are crucial for inner ear development, but whether Id mutations affect middle ear function remains unknown. In this study, we obtained ID1-/-; Id3+/- mice and ID1+/-; Id3-/- mice and carefully examined their middle ear morphology and auditory function. Our study revealed a high incidence (>50%) of middle ear infection in the compound mutant mice. These mutant mice demonstrated hearing impairment starting around 30 days of age, as the mutant mice presented elevated auditory brainstem response (ABR) thresholds compared to those of the littermate controls. The distortion product of otoacoustic emission (DPOAE) was also used to evaluate the conductive function of the middle ear, and we found much lower DPOAE amplitudes in the mutant mice, suggesting sound transduction in the mutant middle ear is compromised. This is the first study of the middle ears of Id compound mutant mice, and high incidence of middle ear infection determined by otoscopy and histological analysis of middle ear suggests that ID1/Id3 compound mutant mice are a novel model for human otitis media (OM).
-
Image_1_Hearing Loss in ID1−/−; Id3+/− and ID1+/−; Id3−/− Mice Is Associated With a High Incidence of Middle Ear Infection (Otitis Media).TIF
'Frontiers Media SA', 2021Co-Authors: Qingyin Zheng, Tihua Zheng, Aizhen Zhang, Bin Yan, Zhaoqiang Zhang, Yan ZhangAbstract:Inhibitors of differentiation/DNA binding (Id) proteins are crucial for inner ear development, but whether Id mutations affect middle ear function remains unknown. In this study, we obtained ID1−/−; Id3+/− mice and ID1+/−; Id3−/− mice and carefully examined their middle ear morphology and auditory function. Our study revealed a high incidence (>50%) of middle ear infection in the compound mutant mice. These mutant mice demonstrated hearing impairment starting around 30 days of age, as the mutant mice presented elevated auditory brainstem response (ABR) thresholds compared to those of the littermate controls. The distortion product of otoacoustic emission (DPOAE) was also used to evaluate the conductive function of the middle ear, and we found much lower DPOAE amplitudes in the mutant mice, suggesting sound transduction in the mutant middle ear is compromised. This is the first study of the middle ears of Id compound mutant mice, and high incidence of middle ear infection determined by otoscopy and histological analysis of middle ear suggests that ID1/Id3 compound mutant mice are a novel model for human otitis media (OM).
Youhua Liu - One of the best experts on this subject based on the ideXlab platform.
-
tubular cell dedifferentiation and peritubular inflammation are coupled by the transcription regulator ID1 in renal fibrogenesis
Kidney International, 2012Co-Authors: Xiaoyan Wen, Youhua LiuAbstract: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.
-
tubular epithelial cell dedifferentiation is driven by the helix loop helix transcriptional inhibitor ID1
Journal of The American Society of Nephrology, 2007Co-Authors: Junwei Yang, Jianhua Luo, Shoukat Dedhar, Youhua LiuAbstract:In the fibrotic kidney, tubular cells undergo epithelial-to-mesenchymal transition (EMT), a phenotypic conversion that is characterized by sequential loss of epithelial markers and gain of mesenchymal features. For understanding of the molecular mechanism that governs this process, a high-throughput gene expression microarray analysis was used to identify the critical genes in the initial phase of the TGF-beta1-mediated EMT. Inhibitor of differentiation-1 (ID1), a dominant negative antagonist of the basic helix-loop-helix transcription factors, was found to be induced rapidly in human proximal tubular epithelial cells after TGF-beta1 treatment. This induction of ID1 depended on intracellular Smad signaling. Ectopic expression of ID1 suppressed epithelial E-cadherin and zonula occludens-1 expression. ID1 physically formed complex with basic helix-loop-helix transcription factor HEB (Hela E-box binding factor), sequestered its ability to bind to E-box, and repressed the trans-activation of E-cadherin promoter. However, overexpression of ID1 failed to induce alpha-smooth muscle actin, matrix metalloproteinase-2, fibronectin, and integrin-linked kinase (ILK), indicating its inability to confer a complete EMT. Overexpression of ILK or inhibition of ILK activity had no effect on ID1 induction by TGF-beta1, suggesting that ID1 and ILK have independent roles in epithelial dedifferentiation and EMT. In vivo, ID1 was induced exclusively in the degenerated, dilated renal tubular epithelium after unilateral ureteral obstruction. These studies identify ID1 transcriptional inhibitor as a crucial player in mediating cell dedifferentiation of renal tubular epithelium and suggest that EMT is a multistep process in which loss of epithelial adhesion does not necessarily lead to an autonomous mesenchymal transition.
Marco Seandel - One of the best experts on this subject based on the ideXlab platform.
-
expansion and maintenance of human embryonic stem cell derived endothelial cells by tgfβ inhibition is ID1 dependent
Nature Biotechnology, 2010Co-Authors: Daylon James, Hyung-song Nam, Daniel J Nolan, Marco Seandel, Tyler Janovitz, Mark J Tomishima, Lorenz Studer, Gabsang LeeAbstract:Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell-specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)beta at day 7 of differentiation increases hVPr-GFP(+) cells by tenfold. In phase 2, TGFbeta inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of ID1(high)VEGFR2(high)VE-cadherin(+) ephrinB2(+). Using an ID1-YFP hESC reporter line, we showed that TGFbeta inhibition sustains ID1 expression in hESC-derived endothelial cells and that ID1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.
-
expansion and maintenance of human embryonic stem cell derived endothelial cells by tgfβ inhibition is ID1 dependent
Nature Biotechnology, 2010Co-Authors: Daylon James, Robert Benezra, Daniel J Nolan, David Lyden, Marco Seandel, Tyler Janovitz, Mark J Tomishima, Lorenz Studer, N ZaninovicAbstract:Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell–specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)β at day 7 of differentiation increases hVPr-GFP+ cells by tenfold. In phase 2, TGFβ inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of ID1highVEGFR2highVE-cadherin+ ephrinB2+. Using an ID1-YFP hESC reporter line, we showed that TGFβ inhibition sustains ID1 expression in hESC-derived endothelial cells and that ID1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.
