Inhibitor Of Differentiation Proteins Research Articles

Abstract 1278: ID3 controlled dynamic Pyk2 localization is important for the mobility of cancer stem cells

Abstract Treatment of brain metastases remains a clinical barrier in breast cancer patients. Secondary breast tumors in the brain are reported to be enriched with breast cancer stem cell (BCSC) markers suggesting that BCSCs infiltrate the brain. Brain infiltration by BCSCs is complex involving detachment from the primary tumor, entering the blood, surviving the microvascular niche, and crossing the blood brain barrier (BBB). It is critical to understand how BCSCs move through the BBB to prevent brain metastases. Nuclear respiratory factor 1 (NRF1) transcription factor, Pyk2 non-receptor tyrosine kinase, and inhibitor of differentiation protein 3 (ID3) have been shown to contribute to the pathogenesis of cancer through inducing CSCs or increasing mobility of these cells. Recently, we showed that NRF1-induced BCSCs crossed the BBB which were guided by ID3 overexpressing endothelial stem cells. In this study, we investigated the NRF1/ID3/Pyk2 axis contributing to BCSCs mobility. NRF1 promoted migration of BCSCs and mesenchymal MDAMB231 cells through brain endothelial cells cultured with astrocytes. ID3 overexpressing endothelial cells promoted adhesion with BCSCs and mobility of BCSCs across the BBB model. In addition, ID3 controlled Pyk2 localization which is important for mobility of stem cells and cancer cells. Endothelial cells were treated with a known risk factors for breast cancer, 17β-estradiol (E2) and environmental pollutant PCB153. Treatment with PCB153 or E2 showed increased growth of BCSCs tumor spheroids and mobility of ESCs. Exosomal ID3 released from endothelial cells also supported the growth of BCSCs and provide the basis for paracrine effects by endothelial stem cells associated with breast tumors. These findings show for the first time a novel role for ID3 and Pyk2 by which endothelial stem cells support breast tumor metastases where they most likely facilitate the colonization, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3/Pyk2 modifying agents to prevent the spread of breast cancer to the brain. Citation Format: Jayanta Das, Alok Deoraj, Deodutta Roy, Quentin Felty. ID3 controlled dynamic Pyk2 localization is important for the mobility of cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1278.

Brain infiltration of breast cancer stem cells is facilitated by paracrine signaling by inhibitor of differentiation 3 to nuclear respiratory factor 1.

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 ESCsID3 was detected. NRF1-induced BCSCs crossed through the BBB model and this was promoted by ESCsID3. We also showed that environmental relevant exposure to PCBs (PCB153 and PCB77) produced differential effects. Treatment with PCB153 showed increased growth of NRF1-induced BCSCs tumor spheroids and increased in vivo migration of ESCsID3. Exosomal ID3 released from endothelial cells also supported the growth of NRF1-induced BCSCs and provide the basis for paracrine effects by ESCsID3 associated with breast tumors. Xenograft experiments showed that ID3 overexpressing brain ESCs not only supported the growth of BCSC tumor spheroids but guided them to the neural crest in zebrafish. These findings show for the first time a novel role for ID3 and NRF1 by which ESCsID3 help guide BCSCsNRF1 to distant metastatic sites where they most likely facilitate the colonization, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3 modifying agents to prevent the spread of breast cancer to the brain.

Heme Oxygenase-1 (HMOX-1) and inhibitor of differentiation proteins (ID1, ID3) are key response mechanisms against iron-overload in pancreatic β-cells

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.

Id Proteins Promote a Cancer Stem Cell Phenotype in Mouse Models of Triple Negative Breast Cancer via Negative Regulation of Robo1.

Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1+ cells. Id1+ cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.

Integrated Chip-Seq and RNA-Seq Data Analysis Coupled with Bioinformatics Approaches to Investigate Regulatory Landscape of Transcription Modulators in Breast Cancer Cells.

The objective of this chapter is to describe step-by-step bioinformatics and functional genomics solutions for analyzing ChIP-Seq and RNA-Seq data for understanding the regulatory mechanisms of chromatin modifiers and transcription factors that can drive pathogenesis of chronic complex human diseases, such as cancer. Here we have used two transcription regulatory proteins: nuclear respiratory factor 1 (NRF1) and inhibitor of differentiation protein 3 (ID3) for ChIP-Seq and RNA-Seq data as examples for discussing the importance of selecting the appropriate computational analysis methods, software, and parameters for the processing of raw data as well as their integrative regulatory landscape analysis to obtain accurate and reliable results. Both ChIP-Seq and RNA-Seq analytic methodologies are used as instructional examples to identify NRF1 or ID3 binding to the promoters and enhancers in the genome and their effects on the activity as well as to discover target genes that can drive breast cancer.

The synergic inhibitory effects of dark tea (Camellia sinensis) extract and p38 inhibition on the growth of pancreatic cancer cells.

Background: Dark tea is one of the most popular types of Chinese tea, which has been reported to exhibit anti-obesity, anti-oxidation and antitumor activities in according human cell lines. In terms of tumorigenesis, the systemic study of the physiological effect of specific fraction of dark tea and the relevant molecular mechanism warrant more attention.Methods: Dark tea was firstly isolated through solvent extraction method. Dissolved ethyl acetate extract was further fractioned by elution with various concentration of ethyl alcohol. The cytotoxicity effect of dark tea on cell proliferation was evaluated by CCK8 assay in HPDE human normal pancreatic duct epithelial cells, SW1990 and PANC-1 human pancreatic cancer cells, and SW1116 human colorectal cancer cells. Immunoblotting and flow cytometry analysis were utilized to examine the status of protein and reactive oxygen species respectively. Gene expression profile was analyzed by cDNA microarray and real-time PCR. The plasmid for ID1 expression was stably transfected into SW1990 cells for relevant functional analysis. The effect of dark tea extract on tumorigenesis in vivo was studied in xenograft tumor model.Results: Water eluate fraction of the ethyl acetate extract from dark tea inhibited the growth of SW1990, PANC-1 and SW1116 cells more efficiently compared with that in HPDE cells. Meanwhile, p38 activity was increased and AKT activity was dropped in cancer cells with dark tea extract treatment. Further functional analyses indicated that water eluate fraction and p38 inhibitor treatment exerted a synergic inhibitory effect on cancer cells growth, which was related to their suppressive effect on expression level of ID1 (inhibitor of differentiation protein 1), which was highly expressed in cancer cells. The analysis utilizing xenograft tumor model further indicated water eluate fraction exhibited a significantly inhibitory effect on tumorigenesis.Conclusion: Based on the sequential extraction procedure, our results reveal the inhibitory effect of water eluate fraction of the ethyl acetate extract from dark tea and its synergistic effect with p38 inhibition on the growth of pancreatic cancer cells, in which ID1 is identified as a downstream effector. This sheds insights into the physiological relevance of specific fraction of dark tea to tumorigenesis in pancreatic cancer.

Contribution of Inhibitor of Differentiation and Estrogenic Endocrine Disruptors to Neurocognitive Disorders.

The devastating growth in the worldwide frequency of neurocognitive disorders and its allied difficulties, such as decline in memory, spatial competency, and ability to focus, poses a significant psychological public health problem. Inhibitor of differentiation (ID) proteins are members of a family of helix-loop-helix (HLH) transcription factors. ID proteins have been demonstrated to be involved in neurodevelopmental and depressive diseases and, thus, may influence neurocognitive deficiencies due to environmental exposure. Previously, it has been demonstrated that environmental factors, such as estrogenic endocrine disruptors (EEDs), have played an essential role in the influence of various neurocognitive disorders such as Alzheimer’s, dementia, and Parkinson’s disease. Based on this increasing number of reports, we consider the impact of these environmental pollutants on ID proteins. Better understanding of how these ID proteins by which EED exposure can affect neurocognitive disorders in populations will prospectively deliver valuable information in the impediment and regulation of these diseases linked with environmental factor exposure.

Abstract 1128: Exosomal ID3 is pro-metastatic through guiding NRF1-induced breast cancer stem cells across the blood-brain-barrier

Abstract Nuclear respiratory factor 1 (NRF1) and inhibitor of differentiation protein 3 (ID3) are transcription regulating proteins. Recently we have uncovered a novel oncogenic function of NRF1 in breast cancer development and progression. In this study, we tested the postulate that exposure to PCB153 contributes in ID3 over-expressing endothelial stem cells (EndSCs) chaperoning and guiding of mesenchymal NRF1 breast cancer stem cells (BCSCs) across the blood-brain barrier. BCSCs/EndSCs were subjected to functional gain/loss of ID3 and/or NRF1 to test if PCB153 [1ng/ml] exposure produces NRF/ID3 signals regulating lineage specific BCSCs organ entry. First, we tested whether NRF1 promotes transmigration of breast cancer using a 3D blood-brain barrier (BBB) model consisting of breast tumor initiating cells (BTICs) or MDAMB231 cells, brain endothelial cell layer, and astrocytes. NRF1 overexpression increased the propensity for BTICs and mesenchymal enriched MDAMB231 BCSCs to adhere to brain endothelial cells and migrate across a human BBB model. Increased adhesion of NRF1+ BCSCs with ID3+ EndSCs was detected. ID3 EndSCs promoted the transmigration of BCSCs through the BBB. We found differential effects by endocrine disruptors, namely PCB153 and PCB77. The treatment with PCB153 showed increased growth of NRF1+ BCSCs tumor spheroids. PCB153 also induced the tumor cell adhesion to microvascular endothelium and transendothelial migration of BCSCs. The exosomal ID3 released from endothelial cells helped the mesenchymal NRF1+ BCSCs to cross through blood brain barriers. Xenograft experiments showed that ID3+ brain EndSCs not only supported the growth of estrogen treated NRF1+ BCSC tumorospheroids, but guided them to the brain in zebrafish. These findings show for the first time a key role for ID3 and NRF1 by which specific circulatory EndSCs accompany a particular sub-type of BCSCs to distant metastatic sites where they most likely facilitate the seeding, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3 modifying agents that prevent the spread of breast cancer to the brain. This work was, in part, supported by a VA MERIT Review (VA BX001463) grant to DR. Citation Format: Jayanta K. Das, Mayur Doke, Alok Deoraj, Quentin Felty, Deodutta Roy. Exosomal ID3 is pro-metastatic through guiding NRF1-induced breast cancer stem cells across the blood-brain-barrier [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1128.

Abstract 4838: Inhibitor of differentiation-1 (Id1) expression correlates with epithelial-mesenchymal transition (EMT)-related proteins in epithelial ovarian cancer (EOC) and constitutes a novel prognostic factor

Abstract Aim The first aim of this work was to investigate the expression of inhibitors of differentiation (ID) proteins in advanced Epithelial Ovarian Cancer (EOC) and its relation with Epithelial to Mesenchymal (EMT)-related markers. Secondly, we also studied their relation with patient clinical and pathological characteristics in order to establish their role as prognostic factors. Methods ID proteins expression was analyzed in 59 samples from EOC patients. We performed inmunohistochemistry (IHC) on tissue microarray sections with specific antibodies for ID1, ID2, ID3 and ID4. Immunoreactivity was H-scored for intensity (range 0 to 3) multiplied by percentage of positive cells. It has been previously described that ID genes are involved in EMT, so we correlated IDs with EMT- regulators, including ECAD, ZEB1, ZEB2, SNAIL, SLUG, LOX and LOXL2. We also explored their relation with clinical variables in our cohort. Results ID2 and ID3 proteins are uniformly expressed in our EOC series. ID1 and ID2 are also overexpressed in different proportions (41% and 88% of cases respectively). No correlation between increased ID proteins expression and histological subtype, tumor grading, debulking surgery status or treatment response was detected. We have found that ID1 overexpression correlates with prognosis, for Overall Survival (OS), HR: 1.05 (95% CI: 1.02 - 1.07) in the univariate analysis and for Time to Treatment Failure (TTF) in both, uni- and multivariate analysis adjusted to clinical factors (HR: 1.06; 95%CI: 1.02 - 1.1). This finding was confirmed when specifically analyzing the worst prognosis group of patients with high grade serous malignancies. ID1 is known to be associated with more invasive features of cancer and with the EMT. In our cohort, its expression is correlated with some EMT-regulators, as ECAD and SLUG (p < 0,005). Conclusions ID proteins expression is frequently deregulated in EOC patients and it seems to influence clinical prognosis, mainly, ID1. Their usefulness as prognostic biomarkers should be further investigated in larger series. Citation Format: Victoria Heredia, Andres Redondo, Jorge Barriuso, Alberto Berjón, Cristian Perna, Patricia Cruz, Alicia Hernández, Javier de Santiago, Esther Díaz, María Miguel, Beatriz Castelo, Laura Yébenes, Jaime Feliú, David Hardisson, Marta Mendiola. Inhibitor of differentiation-1 (Id1) expression correlates with epithelial-mesenchymal transition (EMT)-related proteins in epithelial ovarian cancer (EOC) and constitutes a novel prognostic factor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4838. doi:10.1158/1538-7445.AM2015-4838

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

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. 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. 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. 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.

Abstract 4518: PCL/Maltodextrin delivered ID4 maintains its tumor suppressor role.

Abstract INTRODUCTION: The Inhibitor of differentiation protein (Id) family has generated much attention in cancer cell biology due to its multifaceted role. These proteins have a helix–loop– helix (HLH) domain required for association with transcriptional factors but lack the basic amino acids required for DNA-binding therefore act as dominant-negative regulators of transcription. Tumor suppressor roles have been strongly associated with Id4 one of the four different isoforms identified so far. A recent study by Chaudhary et al indicated that Id4 was a potential tumor suppressor of prostate cancer with ectopic expression of Id4 in DU145 leading to increased apoptosis and decreased cell proliferation due in part by an S-phase arrest. Methylation mediated silencing of the Id4 gene has been associated with development of prostate cancer. In this study we evaluated the pro-apoptotic potential of intracellularly delivered Id4 using PCL/Maltodextrin nanoparticles as the delivery vector. METHOD: PCL/Maltodextrin nanoparticles were formulated using the double emulsion solvent evaporation technique with a Nano Debee high-pressure homogenizer. Nanoparticles were lyophilized and characterized for their size, zeta potential, and encapsulation efficiency. Western blot analysis was conducted on Id4 loaded nanoparticles and nanoparticle treated DU 145 cell lines to confirm the presence and delivery of intact protein. Immuno-cytochemical analysis of Id4 expression was conducted on DU-145, LNCaP and LNCaP cells silenced with Id4 (LNCaP-Id4) pre and post treatment with Id4 loaded nanoparticles.The effect of nanoparticle delivered Id4 on cellular processes such as apoptosis, proliferation and transwell migration was also determined. RESULTS: Id4 loaded PCL/Maltodextrin nanoparticles was successfully formulated with an average particle size of about 200nm. The formulation had encapsulation efficiency of 78.83±7.40% and percentage yield over 98%. Western blot analysis reveals that intact Id4 protein was successfully loaded and delivered to the cells. Immuno-cytochemical analysis of the three cell lines showed an efficient intracellular delivery of Id4 using PL/Maltodextrin with the intracellular amount increasing with time. The ICC analysis also showed possible intra- nuclear localization of Id4. Intracellular delivered Id4 increased the levels of Caspase 3/9 by about three folds associated with decreased mitochondrial membrane potential and plasma membrane asymmetry. The transwell migration assay also showed a significant decrease in cell migration after treatment with Id4 loaded nanoparticles in comparison to the untreated and blank nanoparticle group. CONCLUSION: PCL/Maltodextrin nanocarrier can successfully deliver Id4 to the cytoplasm as well as nucleus of LNCaP-Id4, DU145 and LNCaP. Intracellular delivered Id4 maintains it pro apoptotic potential. Citation Format: Maxwell Korang-Yeboah, Pankaj Sharma, Ashley Evans Knowell, Divya Patel, Yamini Gorantla, Swathi Chinaranagari, Ravi Palaniappa, Jaideep Chaudhary. PCL/Maltodextrin delivered ID4 maintains its tumor suppressor role. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4518. doi:10.1158/1538-7445.AM2013-4518

Inhibitor of differentiation proteins do not influence prognosis of biliary tract cancer

To investigate the expression and clinical relevance of inhibitor of differentiation (ID) proteins in biliary tract cancer. ID protein expression was analyzed in 129 samples from patients with advanced biliary tract cancer (BTC) (45 extrahepatic, 50 intrahepatic, and 34 gallbladder cancers), compared to normal controls and correlated with clinical an pathological parameters. ID1-3 proteins are frequently overexpressed in all BTC subtypes analyzed. No correlation between increased ID protein expression and tumor grading, tumor subtype or treatment response was detected. Survival was influenced primary tumor localization (extrahepatic vs intrahepatic and gall bladder cancer, OS 1.5 years vs 0.9 years vs 0.7 years, P = 0.002), by stage at diagnosis (OS 2.7 years in stage I vs 0.6 years in stage IV, P < 0.001), resection status and response to systemic chemotherapy. In a multivariate model, ID protein expression did not correlate with clinical prognosis. Nevertheless, there was a trend of shorter OS in patients with loss of cytoplasmic ID4 protein expression (P = 0.076). ID protein expression is frequently deregulated in BTC but does not influence clinical prognosis. Their usefulness as prognostic biomarkers in BTC is very limited.

ID family protein expression and regulation in hypoxic pulmonary hypertension

Bone morphogenetic protein (BMP) signaling has been linked to the development of pulmonary hypertension (PH). Inhibitors of differentiation (ID) proteins (ID1-4) are a family of basic helix-loop-helix transcription factors that are downstream targets of the BMP signaling pathway, but the role that ID proteins play in the development of PH is unknown. To address this, we evaluated pulmonary expression of ID proteins in a mouse model of hypoxia-induced PH. There is selective induction of ID1 and ID3 expression in hypoxic pulmonary vascular smooth muscle cells (VSMCs) in vivo, and ID1 and ID3 expression are increased by hypoxia in cultured pulmonary VSMCs in a BMP-dependent fashion. ID4 protein is barely detectable in the mouse lung, and while ID2 is induced in hypoxic peripheral VSMCs in vivo, it is not increased by hypoxia or BMP signaling in cultured pulmonary VSMCs. In addition, the PH response to chronic hypoxia is indistinguishable between wild type and Id1 null mice. This is associated with a compensatory increase in ID3 but not ID2 expression in pulmonary VSMCs of Id1 null mice. These findings indicate that ID1 is dispensable for mounting a normal pulmonary vascular response to hypoxia, but suggest that ID3 may compensate for loss of ID1 expression in pulmonary VSMCs. Taken together, these findings indicate that ID1 and ID3 expression are regulated in a BMP-dependent fashion in hypoxic pulmonary VSMCs, and that ID1 and ID3 may play a cooperative role in regulating BMP-dependent VSMC responses to chronic hypoxia.

Effect of inhibitor of differentiation-1 on murine dendritic cell sarcoma cells

To investigate the effect of down-expression of inhibitor of differentiation-1 (Id-1) on the differentiation of dendritic cell sarcoma (DCS) cells in vitro. Down-regulation of the expression of Id-1 in DCS cells was performed by RNAi, and confirmed by protein and mRNA quantitative analyses. Cellular differentiation and biological behavior including malignant phenotypes of the cells were evaluated. All experiments included negative (no treatment group and no-target siRNA) and positive (induction-differentiation drug sodium butyrate) controls. When the expression of Id-1 was down regulated, the DCS cells showed more mature morphology including cell enlargement, longer cellular extensions, more branches, and decreased nuclear/plasma ratio. Differentiation marker expression (Id-2 and CD86) was also increased. RNAi treated cells at 24 and 48 hours, showed increase percentage of cells at G0/G1 phase and less cells at S phase (P < 0.01). Importantly, the abilities of cell proliferation, colony formation and invasiveness were significantly decreased (P < 0.01), as evidenced by MTT, colony formation and transwell assays respectively. RNAi inhibition of Id-1 protein can induce differentiation of malignant solid tumor cells along with reversion of their malignant phenotype.

Apoptosis and Id2 expression in diaphragm and soleus muscle from the emphysematous hamster

During chronic obstructive pulmonary disease (COPD) diaphragm and peripheral muscle weakness occur. Muscle remodeling and wasting may be a result of apoptosis and changes in muscle-specific transcription factors, such as MyoD, altering muscle-specific gene transcription and muscle regenerative capacity. To investigate this, we instilled under ketamine/xylazine anesthesia porcine elastase in the lungs of hamsters to induce emphysema. The emphysematous hamster is an accepted model for COPD. In the diaphragm and peripheral muscles we assessed the occurrence of apoptosis, and in the diaphragm and soleus also the expression of MyoD and inhibitor of differentiation protein 2 (Id2). There was no significant muscle atrophy in emphysematous hamsters. The mRNA levels of TNF-alpha and markers of apoptosis were significantly elevated in the diaphragm and soleus muscles during emphysema. This was accompanied by an increased presence of nucleosomes in the cytosol. Caspase 3 activity and the DNA-binding activity of the p65 subunit of NF-kappaB, however, were unaltered in all muscles. The protein expression of MyoD and Id2 were decreased and increased in the diaphragm and the soleus muscle, respectively. Thus, despite the absence of muscle atrophy in emphysematous hamsters, there was evidence of increased TNF-alpha expression, apoptosis, and altered muscle-specific transcriptional regulation as reflected by decreased MyoD and elevated Id2 levels at least in the soleus and diaphragm muscle. These alterations may impair the regenerative capacity of skeletal muscles and ultimately contribute to muscle wasting.

Influence of Temperature on the Ontogenetic Expression of Neural Development-Related Genes from Developing Tilapia Brain Expressed Sequence Tags

The developing central neural circuits in teleosts are genetically controlled and temperature-initiated. We compiled a list of transcripts expressed in the developing tilapia (Oreochromis mossambicus) brain using expressed sequence tags derived from the developing brain, and investigated genes with thermosensitive ontogenetic expression. Of 1084 clones, 893 were unique genes, 445 of which were known. Fourteen of the latter were neural development-related, and the ontogenetic expression of nine was temperature-influenced. Discs large homolog 5, myelin expression factor 2, plasticity-related protein-2, tsc2 gene product-related genes, and an inhibitor of differentiation protein 2 (Id2) were differentially temperature-influenced according to their developmental stages. Endothelial differentiation-related factor 1, midkine-related growth factor b, and mitogen-activated protein kinase 14b were specifically influenced by elevated temperature, and beta-catenin-like isoform 1 by lower temperature. Neural development-related genes, particularly those with thermosensitive ontogenetic expression, might be important for developing central neural circuits in teleosts.

Expression of the helix–loop–helix protein inhibitor of DNA binding-1 (ID-1) is activated by all-trans retinoic acid in normal human keratinocytes

The ID (inhibitor of differentiation or DNA binding) helix-loop-helix proteins are important mediators of cellular differentiation and proliferation in a variety of cell types through regulation of gene expression. Overexpression of the ID proteins in normal human keratinocytes results in extension of culture lifespan, indicating that these proteins are important for epidermal differentiation. Our hypothesis is that the ID proteins are targets of the retinoic acid signaling pathway in keratinocytes. Retinoids, vitamin A analogues, are powerful regulators of cell growth and differentiation and are widely used in the prevention and treatment of a variety of cancers in humans. Furthermore, retinoic acid is necessary for the maintenance of epithelial differentiation and demonstrates an inhibitory action on skin carcinogenesis. We examined the effect of all-trans retinoic acid on expression of ID-1, -2, -3, and -4 in normal human keratinocytes and found that exposure of these cells to all-trans retinoic acid causes an increase in both ID-1 and ID-3 gene expression. Furthermore, our data show that this increase is mediated by increased transcription involving several cis-acting elements in the distal portion of the promoter, including a CREB-binding site, an Egr1 element, and an YY1 site. These data demonstrate that the ID proteins are direct targets of the retinoic acid signaling pathway. Given the importance of the ID proteins to epidermal differentiation, these results suggest that IDs may be mediating some of the effects of all-trans retinoic acid in normal human keratinocytes.

The Helix-Loop-Helix Inhibitor of Differentiation (ID) Proteins Induce Post-Mitotic Terminally Differentiated Sertoli Cells to Re-Enter the Cell Cycle and Proliferate

Prior to puberty the Sertoli cells undergo active cell proliferation, and at the onset of puberty they become a terminally differentiated postmitotic cell population that support spermatogenesis. The molecular mechanisms involved in the postmitotic block of pubertal and adult Sertoli cells are unknown. The four known helix-loop-helix ID proteins (i.e., Id1, Id2, Id3, and Id4) are considered dominant negative regulators of cellular differentiation pathways and act as positive regulators of cellular proliferation. ID proteins are expressed at low levels by postpubertal Sertoli cells and are transiently induced by serum. The hypothesis tested was that ID proteins can induce a terminally differentiated postmitotic Sertoli cell to reenter the cell cycle if they are constitutively expressed. To test this hypothesis, ID1 and ID2 were stably integrated and individually overexpressed in postmitotic rat Sertoli cells. Overexpression of ID1 or ID2 allowed postmitotic Sertoli cells to reenter the cell cycle and undergo mitosis. The cells continued to proliferate even after 300 cell doublings. The functional markers of Sertoli cell differentiation such as transferrin, inhibin alpha, Sert1, and androgen binding protein (ABP) continued to be expressed by the proliferating Sertoli cells, but at lower levels. FSH receptor expression was lost in the proliferating Sertoli cell-Id lines. Some Sertoli cell genes, such as cyclic protein 2 (cathepsin L) and Sry-related HMG box protein-11 (Sox11) increase in expression. At no stage of proliferation did the cells exhibit senescence. The expression profile as determined with a microarray protocol of the Sertoli cell-Id lines suggested an overall increase in cell cycle genes and a decrease in growth inhibitory genes. These results demonstrate that overexpression of ID1 and ID2 genes in a postmitotic, terminally differentiated cell type have the capacity to induce reentry into the cell cycle. The observations are discussed in regards to potential future applications in model systems of terminally differentiated cell types such as neurons or myocytes.

Interactions between ID and OLIG proteins mediate the inhibitory effects of BMP4 on oligodendroglial differentiation.

Bone morphogenetic protein (BMP) signaling inhibits the generation of oligodendroglia and enhances generation of astrocytes by neural progenitor cells both in vitro and in vivo. This study examined the mechanisms underlying the effects of BMP signaling on glial lineage commitment. Treatment of cultured neural progenitor cells with BMP4 induced expression of all four members of the inhibitor of differentiation (ID) family of helix-loop-helix transcriptional inhibitors and blocked oligodendrocyte (OL) lineage commitment. Overexpression of Id4 or Id2 but not Id1 or Id3 in cultured progenitor cells reproduced both the inhibitory effects of BMP4 treatment on OL lineage commitment and the stimulatory effects on astrogliogenesis. Conversely, decreasing the levels of Id4 mRNA by RNA interference enhanced OL differentiation and inhibited the effects of BMP4 on glial lineage commitment. This suggests that induction of Id4 expression mediates effects of BMP signaling. Bacterial two-hybrid and co-immunoprecipitation studies demonstrated that ID4, and to a lesser extent ID2, complexed with the basic-helix-loop-helix transcription (bHLH) factors OLIG1 and OLIG2, which are required for the generation of OLs. By contrast, ID1 and ID3 did not complex with the OLIG proteins. In addition, the OLIG and ID proteins both interacted with the E2A proteins E12 and E47. Further, exposure of cultured progenitor cells to BMP4 changed the intracellular localization of OLIG1 and OLIG2 from a predominantly nuclear to a predominantly cytoplasmic localization. These observations suggest that the induction of ID4 and ID2, and their sequestration of both OLIG proteins and E2A proteins mediate the inhibitory effects of BMP signaling on OL lineage commitment and contribute to the generation of astrocytes.