IGFBP-1 Promoter Research Articles

Histone MARylation regulates lipid metabolism in colorectal cancer by promoting IGFBP1 methylation

In the global health community, colorectal cancer (CRC) is a major concern, with a high rate of incidence. Mono-ADP-ribosylation (MARylation) is a type of epigenetics and recognized as one of the causes of CRC development and progression. Although the modification level and target proteins in CRC remain unclear, it has been found that MARylation of arginine-117 of histone 3 (H3R117) promotes the proliferation, upregulates methylation of tumor suppressor gene, and is tightly associated with the metabolic processes in LoVo cells. Lipid metabolism disorder is involved in the development of CRC at the early stage. Our study revealed that MARylation of H3R117 of the LoVo cells modulated lipid metabolism, increased cholesterol synthesis, promoted lipid raft (LR) protein IGF-1R distribution, and inhibited cell apoptosis through IGFBP1. In addition, bioinformatics analyses revealed that IGFBP1 promoter was hypermethylated in CRC when compared to that in normal tissues. Moreover, H3R117 MARylation upregulated the methylation of IGFBP1 promoter through histone H3 citrullination (H3cit) by increasing the H3K9me2, heterochromatin protein1 (HP1), and DNA methyltransferase 1 (DNMT1) enrichment of IGFBP1 promoter. Accordingly, IGFBP1 may function as a tumor suppressor gene, while H3R117 MARylation may promote CRC development. Our study findings enrich the available data on epigenetics of CRC and provide a new idea and experimental basis for H3R117 MARylation as a target in CRC treatment.

The role of HMGA2 in activating the IGFBP2 expression to promote angiogenesis and LUAD metastasis via the PI3K/AKT/VEGFA signaling pathway.

This study investigates the molecular mechanism of HMGA2-mediated regulation of IGFBP2 expression in the PI3K/AKT/VEGFA signaling pathway, which is involved in angiogenesis and LUAD metastasis. Target genes with prognostic implications for LUAD patients were selected using bioinformatics, and previously published literature was referenced to predict the molecular regulatory mechanisms. A549 cells were used for in vitro validation. Cell proliferation and viability were assessed using CCK-8 and EdU assays, while cell migration ability was evaluated using Transwell and wound healing assays. Changes in angiogenesis were examined using an angiogenesis assay. The targeted binding of HMGA2 with the IGFBP2 promoter was confirmed through dual luciferase reporter gene experiments and ChIP assays. In vivo validation was performed using a xenograft mouse model, and changes in angiogenesis and tumor metastasis were observed using western blot, immunofluorescence, and H&E staining. Bioinformatics analysis revealed that HMGA2 was one of the AAGs that differed between normal individuals and LUAD patients and could serve as a critical mRNA for predicting LUAD prognosis. Results from in vitro experiments demonstrated that the expression of the HMGA2 gene was significantly upregulated in LUAD cell lines. Through mediating the expression of IGFBP2, the HMGA2 gene activated the PI3K/AKT/VEGFA signaling pathway, promoting the proliferation, migration, and angiogenesis of A549 cells. In vivo, animal experiments further confirmed that HMGA2 facilitated angiogenesis and the development and metastasis of LUAD through mediating IGFBP2 expression and activating the PI3K/AKT/VEGFA signaling pathway. HMGA2 promotes angiogenesis and healthy growth and metastasis of LUAD by activating the PI3K/AKT/VEGFA signaling pathway by mediating IGFBP2 expression.

Sheep IGFBP2 and IGFBP4 promoter methylation regulates gene expression and hair follicle development

BackgroundHair follicle development is important for the fineness of wool. Our previous methylated DNA immunoprecipitation sequencing showed that IGFBP2 and IGFBP4 were differentially methylated at different development stages of the hair follicle of Super Merino sheep. This study selected these two genes as candidates to uncover the influence of their promoter methylation on hair follicle development. ResultsThe results showed that the expression levels of IGFBP2 and IGFBP4 in the skin of very fine wool Super Merino sheep were higher than that in the very thick wool group, and the difference for IGFBP4 was significant. The total methylation rate of IGFBP2 promoter was extremely low, and no significant difference was found in the skin tissue of the Super Merino sheep at different fineness groups. However, the total methylation rate of IGFBP4 promoter in the very thick group was significantly higher than that in the very fine group, and the total DNA methylation levels of the IGFBP4 were significantly negatively correlated with mRNA expression. Furthermore, the transcription factor prediction results showed 104 and 91 candidate binding sites for transcription factors in the promoter regions of IGFBP2 and IGFBP4, respectively. Among these, JUN, EP300, SP1, PAX6, ETS1, and MYOD1 may be related to hair follicle development. ConclusionsThe total methylation level of IGFBP4 promoter was negatively correlated with its mRNA expression in Super Merino sheep skin tissues with different degrees of fineness, which could be used as an epigenetic marker for wool breeding in Super Merino sheep.How to cite: Tian Y, Du J, Yang X, et al. Sheep IGFBP2 and IGFBP4 promoter methylation regulates gene expression and hair follicle development. Electron J Biotechnol 2022;59. https://doi.org/10.1016/j.ejbt.2022.07.001.

HMGA2 contributes to vascular development and sprouting angiogenesis by promoting IGFBP2 production

Angiogenesis is the process by which new blood vessels form from preexisting vessels and regulates the processes of embryonic development, wound healing and tumorigenesis. HMGA2 is involved in the occurrence of several cancers, but its biological role and the exact downstream genes involved in vascular development and sprouting angiogenesis remain largely unknown. Here, we first found that HMGA2 knockdown in zebrafish embryos resulted in defects of central artery formation. RNA sequencing revealed that IGFBP2 was significantly downregulated by interference with HMGA2, and IGFBP2 overexpression reversed the inhibition of brain vascular development caused by HMGA2 deficiency. In vitro, we further found that HMGA2 knockdown blocked the migration, tube formation and branching of HUVECs. Similarly, IGFBP2 protein overexpression attenuated the impairments induced by HMGA2 deficiency. Moreover, the promotion of angiogenesis by HMGA2 overexpression was verified in a Matrigel plug assay. We next found that HMGA2 bound directly to a region in the IGFBP2 promoter and positively regulated IGFBP2 expression. Interestingly, the mRNA expression levels of HMGA2 and IGFBP2 were increased significantly in the peripheral blood of hemangioma patients, indicating that overexpression of HMGA2 and IGFBP2 results in vessel formation, consistent with the results of the in vivo and in vitro experiments. In summary, our findings demonstrate that HMGA2 promotes central artery formation by modulating angiogenesis via IGFBP2 induction.

Functional annotation of the 2q35 breast cancer risk locus implicates a structural variant in influencing activity of a long-range enhancer element

A combination of genetic and functional approaches has identified three independent breast cancer risk loci at 2q35. A recent fine-scale mapping analysis to refine these associations resulted in 1 (signal 1), 5 (signal 2), and 42 (signal 3) credible causal variants at these loci. We used publicly available in silico DNase I and ChIP-seq data with in vitro reporter gene and CRISPR assays to annotate signals 2 and 3. We identified putative regulatory elements that enhanced cell-type-specific transcription from the IGFBP5 promoter at both signals (30- to 40-fold increased expression by the putative regulatory element at signal 2, 2- to 3-fold by the putative regulatory element at signal 3). We further identified one of the five credible causal variants at signal 2, a 1.4 kb deletion (esv3594306), as the likely causal variant; the deletion allele of this variant was associated with an average additional increase in IGFBP5 expression of 1.3-fold (MCF-7) and 2.2-fold (T-47D). We propose a model in which the deletion allele of esv3594306 juxtaposes two transcription factor binding regions (annotated by estrogen receptor alpha ChIP-seq peaks) to generate a single extended regulatory element. This regulatory element increases cell-type-specific expression of the tumor suppressor gene IGFBP5 and, thereby, reduces risk of estrogen receptor-positive breast cancer (odds ratio = 0.77, 95% CI 0.74–0.81, p = 3.1 × 10−31).

Transcription factor C/EBPβ induces genome-wide H3K27ac and upregulates gene expression during decidualization of human endometrial stromal cells

We previously reported that H3K27 acetylation (H3K27ac) increases throughout the genome during decidualization of human endometrial stromal cells (ESCs). However, its mechanisms have not been clarified. We also reported that C/EBPβ acts as a pioneer factor initiating chromatin remodeling by increasing H3K27ac of IGFBP-1 and PRL promoters. Therefore, C/EBPβ may be involved in the genome-wide increase of H3K27ac during decidualization. In this study, we investigated whether C/EBPβ causes genome-wide H3K27ac modifications and regulates gene expressions during decidualization. cAMP was used to induce decidualization. Three types of cells (control cells, cAMP-treated cells, and cAMP-treated + C/EBPβ-knockdowned cells by siRNA) were generated. Of 4190 genes that were upregulated by cAMP, C/EBPβ knockdown inhibited these upregulation in 2239 genes (53.4%), indicating that they are under the regulation of C/EBPβ. cAMP increased H3K27ac in 1272 of the 2239 genes. C/EBPβ knockdown abolished the increase of H3K27ac in almost all genes (1263 genes, 99.3%), suggesting that C/EBPβ can upregulate gene expression by increasing H3K27ac. To investigate how C/EBPβ regulates H3K27ac throughout the genome, we tested the hypothesis that C/EBPβ binds to its binding regions and recruits cofactors with histone acetyltransferase activities. To do this, we collated our ChIP-sequence data with public ChIP-sequence database of transcription factors, and found that p300 is the most likely cofactor that binds to the H3K27ac-increased-regions with C/EBPβ. ChIP-qPCR of several genes confirmed that C/EBPβ binds to the target regions, recruits p300, and increases H3K27ac. Our genome-wide analysis revealed that C/EBPβ induces H3K27ac throughout the genome and upregulates gene expressions during decidualization by recruiting p300 to the promoters.

Bisphenol a affects endometrial stromal cells decidualization, involvement of epigenetic regulation

Bisphenol A(BPA) is one of the most widespread endocrine disruptors in the environment and is associated with reproductive diseases. In this study, we focused on the correlation between environmentally relevant levels of BPA exposure and histone modification during endometrial stromal cells decidualization. BPA exposure changed the morphology of decidualized endometrial stromal cells, with inhibition of mixed-lineage leukemia 1(MLL1) and induction of enhancer of zeste homolog2 (EZH2) during in vitro decidualization. The expression of HOXA10, PRL and IGFBP-1 was down-regulated upon BPA treatment. Furthermore, chromatin immunoprecipitation quantitative PCR(ChIP-qPCR) was performed to evaluate the recruitment of histone-3, lysine-4 trimethylation (H3K4me3) and histone-3, lysine-27 trimethylation (H3K27me3) at the gene promoters. The decreased H3K4me3 and the increased H3K27me3 at HOXA10, PRL and IGFBP-1 promoter regions were consistent with the expression of MLL1 and EZH2 respectively. The effect of BPA on MLL1 and EZH2 could be abrogated by ICI 182,780. Our study provides the first indication that environmentally relevant levels of BPA exposure can regulate the expression of decidualization-related genes by affecting histone modification, impairing endometrial decidualization.

PoFUT1 promotes endometrial decidualization by enhancing the O-fucosylation of Notch1

BackgroundEndometrial stromal cell decidualization is critical for embryo implantation. Dysfunctional decidualization leads to implantation failure, miscarriage and even pregnancy associated disorders in subsequent pregnancy trimesters. Protein glycosylation is involved in many physiological and pathological processes. Protein O-fucosyltransferase 1 (poFUT1) is the key enzyme for the O-fucosylation of proteins. However, the role and mechanism of poFUT1 in human endometrial stromal cell decidualization remain elusive. MethodsWe employed immunohistochemistry to detect the level of poFUT1 in the uterine endometrium from those of the proliferative phase, secretory phase, early pregnancy women and miscarriage patients. Using human endometrial stromal cells (hESCs) and a mouse model, the underlying mechanisms of poFUT1 in decidualization was investigated. FindingsThe level of poFUT1 was increased in the stromal cells of the secretory phase relative to those in the proliferative phase of the menstrual cycle, and decreased in the stromal cells of miscarriage patients compared to women with healthy early pregnancies. Furthermore, we found that poFUT1 promoted hESCs decidualization. The results also demonstrated that poFUT1 increased O-fucosylation on Notch1 in hESCs, which activated Notch1 signaling pathway. Activated Notch1 (NICD), as a specific trans-factor of PRL and IGFBP1 promoters, enhanced PRL and IGFBP1 transcriptional activity, thus inducing hESCs decidualization. InterpretationLevel of poFUT1 is lower in the uterine endometrium from miscarriage patients than early pregnancy women. poFUT1 is critical in endometrial decidualization by controlling the O-fucosylation on Notch1. Our findings provide a new mechanism perspective on poFUT1 in uterine decidualization that may be a useful diagnostic and therapeutic target for miscarriage. FundNational Natural Science Foundation of China (31770857, 31670810 and 31870794). Liaoning Provincial Program for Top Discipline of Basic Medical Sciences.

Depletion of PRDM9 enhances proliferation, migration and chemotaxis potentials in human periodontal ligament stem cells

ABSTRACT Purpose Periodontal ligament mesenchymal stem cells (PDLSCs) are important for periodontal tissue regeneration, but how these cells are regulated remains unclear. PRDM (PRDI-BF1 and RIZ homology domain containing) genes play key roles in cell proliferation and differentiation. The present study aimed to investigate the role of one PRDM gene, PRDM9, in the proliferation, migration and chemotaxis potential of PDLSCs. Materials and methods Cell proliferation was examined on the basis of the cell doubling time, cell counting kit-8 (CCK8) assays, and flow cytometry analysis of the cell cycle. Gene expression was detected by Western blotting and real-time RT-PCR. Scratch migration and Transwell chemotaxis assays were used to analyse cell migration and chemotaxis abilities. Microarray analysis and ChIP assays were used to examine the downstream genes of PRDM9 and the corresponding mechanism. Results The results showed that knock-down of PRDM9 enhanced cell proliferation by promoting cell cycle progression and rapid transition from the G1 to S phase via downregulation of p21 and p27 and upregulation of cyclin E. Additionally, depletion of PRDM9 increased the migration and chemotaxis potential of PDLSCs. Microarray results showed that 13 genes, including IGFBP5, IFI44L, and POSTN, were upregulated and 34 genes, including PIP, were downregulated after the depletion of PRDM9. Furthermore, we observed that the depletion of PRDM9 promoted the transcription of IGFBP5 by increasing H3K4me3 methylation in the IGFBP5 promoter. Conclusion These discoveries indicated that depletion of PRDM9 increased the cell proliferation, migration and chemotaxis potential of PDLSCs and revealed important downstream genes.

PoFUT1 Promotes Endometrial Decidualization by Enhancing the O-Fucosylation of Notch1

Uterine stromal cell decidualization is critical for embryo implantation. Dysfunctional decidualization leads to implantation failure or miscarriage and even initiates pregnancy associated disorders in subsequent pregnancy trimesters. Protein glycosylation is involved in many physiological and pathological processes. Protein O-fucosyltransferase 1 is the key enzyme for the O-fucosylation of proteins. However, the role and mechanism of poFUT1 in hESC decidualization remain elusive. In the current study, we reported that the level of poFUT1 increased in stromal cells in the secretary phase relative to those in the proliferative phase of the menstrual cycle and decreased in the stromal cells of miscarriage patients relative to women with healthy early pregnancies. Using hESC lines and a mouse model, we found that poFUT1 promoted hESC decidualization. We also demonstrated that poFUT1 increased O-fucosylation on Notch1 in hESC, which further activated the Notch1 signalling pathway. Activated Notch1 (NICD) is a specific transfactor of the PRL and IGFBP1 promoters and may increase PRL and IGFBP1 transcriptional activity, further inducing hESC decidualization. Taken together, our findings provide a new mechanistic perspective on poFUT1 in uterine decidualization that may be a useful diagnostic and therapeutic target for miscarriage. Funding Statement: This work was supported by the National Natural Science Foundation of China (No: 31670810 and 31770857). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: This study was approved by the Clinical Ethics Review Board of Dalian Medical University.

The combination of everolimus and terameprocol exerts synergistic antiproliferative effects in endometrial cancer: molecular role of insulin-like growth factor binding protein 2.

Oncogenic PIK3CA mutations are common in endometrial cancers, and the PI3K/AKT/mTOR pathway is targetable by drugs. We sought to investigate whether the combination of an mTOR inhibitor, everolimus (RAD001), and an AKT inhibitor, terameprocol (M4N), exerts better antiproliferative effects in endometrial cancer. The molecular mechanisms underlying their pharmacological action were also examined. The combination of RAD001 and M4N exerted in vitro synergistic effects on cell viability, apoptosis, and expression of IGFBP2 in endometrial cancer cells. Mechanistically, the Sp1 site on the IGFBP2 promoter was required for RAD001- and M4N-induced downregulation. IGFBP2 protein expression was higher in endometrial cancer than in the normal endometrium (P < 0.001). Furthermore, elevated IGFBP2 histoscores were significantly associated with a lower overall survival (P = 0.021). In conclusion, our in vitro results demonstrate that RAD001 and M4N exert synergistic antiproliferative effects against endometrial cancer cells, which appeared to be mediated by the inhibition of IGFBP2, a key anti-apoptotic regulator. Further clinical studies of this drug combination in patients with endometrial cancer may be warranted, especially in the presence of PIK3CA and IGFBP2 aberrations. KEY MESSAGES: RAD001 and M4N synergistically suppress endometrial cancer growth. IGFBP2 is overexpressed in endometrial cancer. The combination of RAD001 and M4N significantly reduces IGFBP2 overexpression. Sp1 binding site on the IGFBP2 promoter is required for RAD001- and M4N-induced downregulation. High IGFBP2 histoscore in endometrial cancer portends a poor prognosis.

FoxO1 and FoxA1/2 form a complex on DNA and cooperate to open chromatin at insulin-regulated genes.

We have previously shown that cooperative, interdependent binding by the pioneer factors FoxO1 and FoxA1/2 is required for recruitment of RNA polymerase II and H3K27 acetylation to the promoters of insulin-regulated genes. However, the underlying mechanisms are unknown. In this study, we demonstrate that, in HepG2 cells, FoxO1 and FoxA2 form a complex on DNA that is disrupted by insulin treatment. Insulin-mediated phosphorylation of FoxO1 and FoxA2 does not impair their cooperative binding to mononucleosome particles assembled from the IGFBP1 promoter, indicating that direct disruption of complex formation by phosphorylation is not responsible for the loss of interdependent FoxO1:FoxA1/2 binding following insulin treatment. Since FoxO1 and FoxA1/2 binding is required for the establishment and maintenance of transcriptionally active chromatin at insulin-regulated genes, we hypothesized that cooperative FoxO1 and FoxA1/2 binding dictates the chromatin remodeling events required for the initial activation of these genes. In support of this idea, we demonstrate that FoxO1 and FoxA2 cooperatively open linker histone compacted chromatin templates containing the IGFBP1 promoter. Taken together, these results provide a mechanism for how interdependent FoxO1:FoxA1/2 binding is negatively impacted by insulin and provide a developmental context for cooperative gene activation by these factors.

The Wilms tumor protein WT1 stimulates transcription of the gene encoding insulin-like growth factor binding protein 5 (IGFBP5)

Insulin-like growth factor (IGF) binding proteins (IGFBPs) constitute a family of six secreted proteins that regulate the signaling of insulin-like growth factors (IGFs). IGFBP5 is the most conserved family member in vertebrates and the major IGF binding protein in bone. IGFBP5 is required for normal development of the musculoskeletal system, and various types of cancer frequently express high levels of IGFP5. Here we identify the gene encoding IGFBP5 as a novel downstream target of the Wilms tumor protein WT1. IGFBP5 and WT1 are expressed in an overlapping pattern in the condensing metanephric mesenchyme of embryonic murine kidneys. Down-regulation of WT1 by transfection with antisense vivo-morpholino significantly decreased Igfbp5 transcripts in murine embryonic kidney explants. Likewise, silencing of Wt1 in a mouse mesonephros-derived cell line reduced Igfbp5 mRNA levels by approximately 80%. Conversely, induction of the WT1(−KTS) isoform, whose role as transcriptional regulator has been firmly established, significantly increased IGFBP5 mRNA and protein levels in osteosarcoma cells. IGFBP5 expression was not significantly changed by WT1(+KTS) protein, which exhibits lower DNA binding affinity than the WT1(−KTS) isoform and has a presumed role in post-transcriptional gene regulation. Luciferase reporter constructs harboring 0.8 and 1.6 kilobases of the murine Igfbp5 promoter, respectively, were stimulated approximately 5-fold by co-transfection of WT1(−KTS). The WT1(+KTS) variant had no significant effect on IGFBP5 promoter activity. Binding of WT1(−KTS), but not of WT1(+KTS) protein, to the IGFBP5 promoter in human osteosarcoma cells was proven by chromatin immunoprecipitation (ChIP) and confirmed by electrophoretic mobility shift assay. These findings demonstrate that WT1 activates transcription of the IGFBP5 gene with possible implications for kidney development and bone (patho)physiology.

A functional variant rs4442975 modulating FOXA1-binding affinity does not influence the risk or progression of breast cancer in Chinese Han population.

The DNA-binding protein FOXA1 has been shown to regulate nearly all estrogen receptor-chromatin interactions, thereby influencing target gene expression levels in breast cancer (BC) cells. Recently, the rs4442975 T-allele, which disrupts the recruitment of FOXA1 and interacts with the IGFBP5 promoter, was associated to BC susceptibility in a European population. We conducted a hospital-based case-control study that included 1227 cases and 1285 controls to explore the potential association between rs4442975 and BC risk in Chinese Han population, and the effect of this SNP on BC progression was also observed in cases. No significant associations between rs4442975 and BC risk were observed under any genetic models, with an odds ratio of 0.96 (95% confidence interval = 0.81-1.15) under the additive model. When stratified based on estrogen or progesterone receptor expression, smoking or drinking habits, or menopausal status, similar negative associations were observed for all subgroups. No significant associations were observed between rs4442975 and traditional progression factors such as tumor size, nodal status, distant metastasis, or TNM staging. These results reveal that rs4442975 may not confer a risk of BC occurrence or progression in the Chinese Han population, which indicates a distinct association related to genetic heterogeneity across ethnic populations.

An intergenic risk locus containing an enhancer deletion in 2q35 modulates breast cancer risk by deregulating IGFBP5 expression.

Breast cancer is the most diagnosed malignancy and the second leading cause of cancer mortality in females. Previous association studies have identified variants on 2q35 associated with the risk of breast cancer. To identify functional susceptibility loci for breast cancer, we interrogated the 2q35 gene desert for chromatin architecture and functional variation correlated with gene expression. We report a novel intergenic breast cancer risk locus containing an enhancer copy number variation (enCNV; deletion) located approximately 400Kb upstream to IGFBP5, which overlaps an intergenic ERα-bound enhancer that loops to the IGFBP5 promoter. The enCNV is correlated with modified ERα binding and monoallelic-repression of IGFBP5 following oestrogen treatment. We investigated the association of enCNV genotype with breast cancer in 1,182 cases and 1,362 controls, and replicate our findings in an independent set of 62,533 cases and 60,966 controls from 41 case control studies and 11 GWAS. We report a dose-dependent inverse association of 2q35 enCNV genotype (percopy OR = 0.68 95%CI 0.55-0.83, P = 0.0002; replication OR = 0.77 95% CI 0.73-0.82, P = 2.1 × 10-19) and identify 13 additional linked variants (r2 > 0.8) in the 20Kb linkage block containing the enCNV (P = 3.2 × 10-15 - 5.6 × 10-17). These associations were independent of previously reported 2q35 variants, rs13387042/rs4442975 and rs16857609, and were stronger for ER-positive than ER-negative disease. Together, these results suggest that 2q35 breast cancer risk loci may be mediating their effect through IGFBP5.

Body size, physical activity, early-life energy restriction, and associations with methylated insulin-like growth factor-binding protein genes in colorectal cancer.

We investigated body size, physical activity, and early-life energy restriction in relation to colorectal tumors with and without methylated insulin-like growth factor-binding protein (IGFBP) genes, which are putative tumor-suppressor genes. We determined IGFBP2, IGFBP3, and IGFBP7 promoter CpG island hypermethylation in tumors of 733 colorectal cancer cases from the Netherlands Cohort Study (N = 120,852). Participants self-reported lifestyle and dietary factors at baseline in 1986. Using a case-cohort approach (N subcohort = 5,000), we estimated hazard ratios (HR) for colorectal cancer by extent of IGFBP methylation. Comparison of the highest versus lowest sex-specific tertiles of adult body mass index (BMI) gave multivariable-adjusted HRs [95% confidence intervals (CI)] for colorectal cancers with 0 (18.7%), 1 (29.5%), 2 (32.4%), and 3 (19.5%) methylated genes of 1.39 (0.88-2.19), 1.11 (0.77-1.62), 1.67 (1.17-2.38), and 2.07 (1.29-3.33), respectively. Other anthropometric measures and physical activity were not associated with colorectal cancer risk by extent of IGFBP methylation, except height in sex-specific analyses for women. Exposure to energy restriction during the Dutch Hunger Winter versus nonexposure gave HRs (95% CIs) for colorectal cancers with 0, 1, 2, and 3 methylated genes of 1.01 (0.67-1.53), 1.03 (0.74-1.44), 0.72 (0.52-0.99), and 0.50 (0.32-0.78), respectively. Adult BMI, height (in women only), and early-life energy restriction were associated with the risk of having a colorectal tumor characterized by IGFBP methylation. Body size may particularly increase the risk of IGFBP gene-methylated colorectal tumors; this finding might facilitate more targeted approaches to prevent obesity-related colorectal cancers.

The PRB-dependent FOXO1/IGFBP-1 axis is essential for progestin to inhibit endometrial epithelial growth

Progestin inhibits the growth of normal and cancerous endometria via the progesterone receptor (PR), but the distinct functions and signalings of PR subtypes have not been fully understood. The aim of the present study was to dissect the key pathways of progestin to inhibit endometrial epithelial growth. Immortalized endometrial epithelial cells (EM-E6/E7/TERT) with stable PRA or PRB expression were established and used for the experiments. In vitro growth inhibition by progestin was mainly observed in EM-E6/E7/TERT cells with PRB rather than those with PRA. RT-PCR assay confirmed that FOXO1, a key gene for progestin action, was up-regulated by progestin in a PRB-dependent manner. cDNA microarray analysis identified IGFBP-1, which contains FOXO1 binding sites on its promoter, to be induced by medroxyprogesterone acetate (MPA) in EM-E6/E7/TERT cells with PRB but not with PRA. siRNA knockdown of FOXO1 disturbed the induction of IGFBP-1 by MPA, while IGFBP-1 knockdown showed no effect on MPA-induced FOXO1 expression, indicating that FOXO1 is an upstream regulator of IGFBP-1. Luciferase reporter assays showed that MPA activated the IGFBP-1 promoter, which was cancelled by FOXO1 knockdown. Chromatin immunoprecipitation assay confirmed the in vivo binding of FOXO1 to the core promoter of IGFBP-1. IGFBP-1 knockdown significantly attenuated the growth inhibitory effects of MPA. The FOXO1/IGFBP-1 axis is essential for PRB-dependent growth inhibition of endometrial epithelial cells, offering a potential therapeutic clue to enhance the progestin effect.

PA1 Protein, a New Competitive Decelerator Acting at More than One Step to Impede Glucocorticoid Receptor-mediated Transactivation

Numerous cofactors modulate the gene regulatory activity of glucocorticoid receptors (GRs) by affecting one or more of the following three major transcriptional properties: the maximal activity of agonists (A(max)), the potency of agonists (EC(50)), and the partial agonist activity of antisteroids (PAA). Here, we report that the recently described nuclear protein, Pax2 transactivation domain interaction protein (PTIP)-associated protein 1 (PA1), is a new inhibitor of GR transactivation. PA1 suppresses A(max), increases the EC(50), and reduces the PAA of an exogenous reporter gene in a manner that is independent of associated PTIP. PA1 is fully active with, and strongly binds to, the C-terminal half of GR. PA1 reverses the effects of the coactivator TIF2 on GR-mediated gene induction but is unable to augment the actions of the corepressor SMRT. Analysis of competition assays between PA1 and TIF2 with an exogenous reporter indicates that the kinetic definition of PA1 action is a competitive decelerator at two sites upstream from where TIF2 acts. With the endogenous genes IGFBP1 and IP6K3, PA1 also represses GR induction, increases the EC(50), and decreases the PAA. ChIP and re-ChIP experiments indicate that PA1 accomplishes this inhibition of the two genes via different mechanisms as follows: PA1 appears to increase GR dissociation from and reduce GR transactivation at the IGFBP1 promoter regions but blocks GR binding to the IP6K3 promoter. We conclude that PA1 is a new competitive decelerator of GR transactivation and can act at more than one molecularly defined step in a manner that depends upon the specific gene.

Growth hormone receptor d3-variant, insulin-like growth factor binding protein-1 –575G/A polymorphism and postnatal catch-up growth: Association with parameters of glucose homeostasis in former extremely low birth weight preterm infants

Low birth weight predisposes to the development of insulin resistance. In addition to auxological parameters such as rapid catch-up growth, low IGFBP-1 serum levels in childhood have been linked to an increased risk of insulin resistance later in life. Concerning postnatal growth, we previously reported the GHRd3-variant to be associated with catch-up growth in preterm infants. In children born small for gestational age, a common IGFBP-1 promoter polymorphism -575G/A has been linked to IGFBP-1 serum levels and has been suggested to be an additional player in the interaction between the IGF-IGFBP-axis and metabolism. We analyzed postnatal growth, metabolic parameters, and genotypes for the GHRd3-variant and IGFBP-1 -575G/A in 51 former extremely low birth weight preterm infants (mean age 5.9 years). GHRd3 but not IGFBP-1 -575G/A was significantly associated with postnatal growth velocity. Catch-up growth, GHRd3, and IFGBP-1 -575G/A did not influence fasting insulin or HOMA-IR. However, we found significantly higher HbA1c and lower IGFBP-1 concentrations in GHRd3-carriers, a finding not seen with respect to IGFBP-1 -575G/A. Interestingly, HbA1c and IGFBP-1 levels also did not differ between children either with or without catch-up growth. In addition to an association with catch-up growth, GHR exon 3 genotype significantly modulates HbA1c and IGFBP-1 concentrations in former ELBW infants. In order to confirm this observation and to clarify whether the GHRd3-variant might be considered as an independent modulator of the low birth weight infant's risk to develop insulin resistance later in life, larger studies extending to later ages are required.

Neuroendocrine Cancer-Specific Up-Regulating Mechanism of Insulin-Like Growth Factor Binding Protein-2 in Small Cell Lung Cancer

Small cell lung cancer (SCLC) exhibits insulin-like growth factor-dependent growth. SCLC is the most aggressive among known in vivo lung cancers, whereas in vitro growth of SCLC is paradoxically slow as compared with that of non-SCLC (NSCLC). In this study, we demonstrate that SCLC cells overexpress insulin-like growth factor binding protein (IGFBP)-2 via NeuroD, a neuroendocrine cell-specific transcription factor. Chromatin immunoprecipitation, electrophoretic mobility shift, and IGFBP-2 promoter assays all revealed that NeuroD binds to the E-box in the 5'-untranslated region of IGFBP-2. A NeuroD transgene in both airway epithelial and NSCLC cells up-regulated the transcription of IGFBP-2 and retarded cell growth. Recombinant IGFBP-2 repressed the growth of both airway epithelial and NSCLC cells in a dose-dependent manner. A NeuroD-specific small interfering RNA repressed IGFBP-2 expression in SCLC, and neutralization of IGFBP-2 and an IGFBP-2-specific small interfering RNA increased SCLC cell growth. Pathological samples of SCLC also expressed IGFBP-2 abundantly, as compared with NSCLC, and showed only rare (8%) IGFBP-2 promoter methylation, whereas the IGFBP-2 promoter was methylated in 71% of adenocarcinomas and 29% of squamous cell carcinomas. These findings suggest that 1) SCLC has an IGFBP-2 overexpression mechanism distinct from NSCLC, 2) secreted IGFBP-2 contributes to the slow growth of SCLC in vitro, and 3) the epigenetic alterations in the IGFBP-2 promoter contribute to the striking differences in IGFBP-2 expression between SCLC and NSCLC in vivo.