Expression analysis of the NDRG2 gene in mouse embryonic and adult tissues

N-myc downstream-regulated gene 2 (NDRG2) is believed to be involved in cell growth events. However, its exact function is still unknown. To elucidate the role of this gene, we used an anti-Ndrg2 monoclonal antibody in immunohistochemistry and immunofluorescence assays to analyze the expression pattern of Ndrg2 protein in mouse embryos at various gestational ages and in a variety of adult mouse tissues. Ndrg2 immunoreactivity was generally localized to the cytoplasm. During mouse development, Ndrg2 expression was observed in many developing tissues and organs including the heart, brain, lung, gut, liver, kidney, skeletal muscle, cartilage, chorion, epidermis, and whisker follicles. Ndrg2 expression was developmentally dynamic, being generally lower in the early stages of development and markedly increasing during later stages. Ndrg2 expression was also observed in a variety of adult mouse tissues, particularly in the heart and brain. This is the first demonstration of Ndrg2 protein expression in both embryonic and adult mouse tissues. Our results suggest that NDRG2 plays important roles in histogenesis and organogenesis.This study was supported by grants from the National Key Basic Research and Development Program (no. 2002CB513007), the National Natural Science Foundation of China (nos. 30370315 and 30171044) and PCSIRT04-59.     

NDRG2在人胚胎呼吸系统的表达特点

目的该实验通过对16-28周人胚胎呼吸系统NDRG2表达的研究,旨在阐明NDRG2在16-28周人胚胎呼吸系统中的表达规律,为进一步明确新的发育相关基因ndrg2的功能提供依据。方法搜集16-28周胎儿呼吸系统的肺和气管组织,制成石蜡切片,用抗NDRG2单克隆抗体,行免疫组化染色(ABC法),从蛋白质水平观察NDRG2表达情况。统计阳性细胞数,利用统计学方法,判断不同胎龄的肺和气管间NDRG2表达有无差别。结果免疫组化染色表明,NDRG2在16-28周胚胎呼吸系统中有广泛的表达,阳性产物主要位于上皮细胞的胞浆中,但是不同胎龄之间NDRG2的表达未见显著差别。结论NDRG2在16-28周胚胎呼吸系统中有广泛的表达,提示NDRG2在早期胚胎的呼吸系统上皮细胞的生长与发育过程中起一定作用。而阳性产物主要表达在上皮细胞的胞浆中,说明NDRG2可能是一种胞浆蛋白。     

NDRG2 is a new HIF-1 target gene necessary for hypoxia-induced apoptosis in A549 cells.

The NDRG2 gene belongs to a family of N-Myc downstream-regulated genes (NDRGs) and is expressed in many normal tissues. NDRG2 gene expression has been shown to be regulated in the stress response of certain cells. However, its function is not yet fully understood. Many studies have demonstrated that hypoxia, one of the stress responses, induced apoptosis in several cell types. In the current study, we investigated NDRG2 involvement in hypoxia response and found that NDRG2 expression was markedly up-regulated in several tumor cell lines exposed to hypoxic conditions or similar stresses at the mRNA and protein level. We also observed that the expression of NDRG2 was regulated by Hypoxia-inducible factor 1 (HIF-1) in tumor cells under hypoxia. Three hypoxia-responsive elements (HREs) in the NDRG2 promoter were identified. HRE1 could directly bind Hif-1 in vivo. Importantly, we found that silencing or enforcing the expression of NDRG2 could strongly inhibit or increase apoptosis. In addition, our data also showed that Ndrg2 was able to be translocated from the cytoplasm to the nucleus, and the segment from 101 to 178 amino acids of Ndrg2 is responsible for its translocation. Taken together, this study suggests that NDRG2 is a Hif-1 target gene and closely related with hypoxia-induced apoptosis in A549 cells.     

N-myc downstream-regulated gene 2 controls astrocyte morphology via Rho-GTPase signaling

Astrocyte undergoes morphology changes that are closely associated with the signaling communications at synapses. N-myc downstream-regulated gene 2 (NDRG2) is specifically expressed in astrocytes and is associated with several important astrocyte functions, but its potential role(s) relating to astrocyte morphological changes remain unknown. Here, primary astrocytes were prepared from neonatal Ndrg2^+/+ and Ndrg2^−/− pups, and the drug Y27632 was used to induce stellation. We then used a variety of methods to measure the levels of NDRG2, α-Actinin4, and glial fibrillary acidic protein (GFAP), and the activity of RhoA, Rac1, and Cdc42 in Y27632-treated astrocytes as well as in Ndrg2^+/+, Ndrg2^−/−, or Ndrg2^−/− + lentivirus (restore NDRG2 expression) astrocytes. We also conducted live-imaging and proteomics studies of the cultured astrocytes. We found that induction of astrocytes stellation (characterized by cytoplasmic retraction and process outgrowth) resulted in increased NDRG2 protein expression and Rac1 activity and in reduced α-Actinin4 protein expression and RhoA activity. Ndrg2 deletion induced astrocyte flattening, whereas the restoration of NDRG2 expression induced stellation. Ndrg2 deletion also significantly increased α-Actinin4 protein expression and RhoA activity yet reduced GFAP protein expression and Rac1 activity, and these trends were reversed by restoration of NDRG2 expression. Collectively, our results showed that Ndrg2 deletion promoted cell proliferation, interrupted stellation capability, and extensively altered the protein expression profiles of proteins that function in Rho-GTPase signaling. These findings suggest that NDRG2 functions to regulate astrocytes morphology via altering the accumulation of the Rho-GTPase signaling pathway components, thereby supporting that NDRG2 should be understood as a regulator of synaptic plasticity and thus neuronal communications.     

Differential expression patterns of NDRG family proteins in the central nervous system.

The N-myc downstream-regulated gene (NDRG) family consists of four proteins: NDRG1, NDRG2, NDRG3, and NDRG4 in mammals. NDRG1 has been thoroughly studied as an intracellular protein associated with stress response, cell growth, and differentiation. A nonsense mutation in the NDRG1 gene causes hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease type 4D. We previously generated Ndrg1-deficient mice and found that they exhibited peripheral nerve degeneration caused by severe demyelination, but that the complicated motor abilities were retained. These results implied that other NDRG family proteins may compensate for the NDRG1 deficiency in the central nervous system. In this study we raised specific antibodies against each member of the NDRG protein family and examined their cellular expression patterns in the mouse brain. In the cerebrum, NDRG1 and NDRG2 were localized in oligodendrocytes and astrocytes, respectively, whereas NDRG3 and NDRG4 were ubiquitous. In the cerebellum, NDRG1 and NDRG4 were localized in Purkinje cells and NDRG2 in Bergmann glial cells. NDRG3 was detected in the nuclei in most cells. These expression patterns demonstrated the cell type-specific and ubiquitous localization of the NDRG family proteins. Each NDRG may play a partially redundant role in specific cells in the brain.     

Association of NDRG1 Gene Promoter Methylation with Reduced NDRG1 Expression in Gastric Cancer Cells and Tissue Specimens

NDRG1 (N-myc downstream-regulated gene 1) plays a role in cell differentiation and suppression of tumor metastasis. This study aims to determine the expression of NDRG1 mRNA and protein in gastric cancer cell lines and tissue specimens and then assess the possible cause of its aberrant expression. Six gastric cancer cell lines and 20 pairs of normal and gastric cancer tissue samples were used to assess NDRG1 expression using Real-time PCR and Western blot. High-resolution melting analysis (HRM) and methylation-specific PCR (MSP) were performed to detect gene mutation and methylation, respectively, in cell lines and tissues samples. Expression of NDRG1 mRNA and protein was downregulated in gastric cancer cell lines and tissues. Specifically, expression of NDRG1 mRNA and protein was lower in all six gastric cancer cell lines than that of normal gastric cells, while 15 out of 20 cases of gastric cancer tissues had the reduced levels of NDRG1 mRNA and protein. HRM data showed that there was no mutation in NDRG1 gene, but MSP data showed high levels of NDRG1 gene promoter methylation in the CpG islands in both cell lines and tissue samples. Moreover, treatment with the DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine upregulated NDRG1 expression in gastric cancer HGC27 cells, but not in the histone deacetylase inhibitor trichostatin A-treated HGC27 cells. In conclusion, this study has shown that expression of NDRG1 mRNA and protein was reduced in gastric cancer cell lines and tissues, which is due to methylation of NDRG1 gene promoter. Further study will unearth the clinical significance of the reduced NDRG1 protein in gastric cancer.     

人NDRG2启动子的克隆与活性鉴定

目的:克隆人NDRG2的启动子,并进行启动子的活性鉴定。方法:用Advantage-GC Taq酶,采用PCR方法,以BCA克隆R-998D1为模板,克隆人NDRG2(-1455/ 274)的启动子。分别构建NDRG2(-1131/ 274)、NDRG2(-273/ 274)、NDRG2(-135/ 274)、NDRG2(-79/ 274)和NDRG2(-79/ 57)等不同长度的截短体,并分别亚克隆入pGL3-basic报告基因载体,测序鉴定。分别转染HEK293和HeLa细胞后,运用双荧光报告基因系统进行启动子活性分析,从而判断核心启动子的位置。结果:人NDRG2的启动子克隆成功,并构建了不同长度的截短体,DNA测序结果与报道一致。报告基因分析结果将人NDRG2启动子的核心区域定位于NDRG2(-79/ 57)。结论:随着人NDRG2的启动子的长度逐渐被截短,启动子的基础活性也逐渐降低,可将人NDRG2启动子的核心区域定位于NDRG2(-79/ 57)。     

The regulation of NDRG2 expression during ATLL development after HTLV-1 infection

N-myc downstream-regulated gene 2 (NDRG2) is a candidate tumor suppressor that is frequently downregulated in adult T-cell leukemia/lymphoma (ATLL) and functions to negatively regulate several cellular signaling pathways as PP2A recruiter. To clarify the molecular mechanisms of suppression of NDRG2 expression, we initially determined the expression pattern of NDRG2 in various types of T-cells and ATLL cells. NDRG2 expression was significantly upregulated in HTLV-1/Tax-immortalized T-cells, which was mediated by NF-κB activation through Tax expression. On the other hand, NDRG2 expression was suppressed in HTLV-1-infected cell lines and various types of ATLL cells, which was dependent on the DNA methylation of the NDRG2 promoter. We found that the expression of enhancer of zeste homolog 2 (EZH2), a member of the polycomb family, is increased in ATLL, and that EZH2 directly binds to the NDRG2 promoter and induces DNA methylation of the NDRG2 promoter. Since the expression of EZH2 were anti-parallelly regulated with the NDRG2 expression, EZH2 might be one of the most important regulators of the downregulation of NDRG2, contributing to enhanced activation of signaling pathways during ATLL development.     

The physical and functional interaction of NDRG2 with MSP58 in cells

NDRG2, a member of N-Myc downstream regulated gene family, exerts the important functions in cell differentiation and tumor suppression. Although the ectopic expressed Ndrg2 inhibits the proliferation of tumor cells, its intracellular signal transduction pathway is hardly known. Here, we identified MSP58, a 58-kDa microspherule protein, as an interacting partner of human Ndrg2 by using yeast two-hybrid screening. The interaction was confirmed by glutathione S-transferase pull-down assay in vitro and by co-immune-precipitation assay in vivo. The forkhead associated domain of MSP58 is essential for its interaction with Ndrg2. Ndrg2 could co-localize with MSP58 in nuclear of HeLa cell during cell stress. Furthermore, the modulation of Ndrg2 level influences the cell cycle process together with MSP58. In conclusion, the findings offered a novel insight into the physiological roles of Ndrg2.     

Cloning and expression pattern of the human NDRG3 gene

We report the cloning and expression pattern of a novel N-myc downstream-regulated gene 3 (NDRG3), located on human chromosome 20q11.21-11.23. The NDRG3 cDNA is 2588 base pair in length, encoding a 363 amino acid polypeptide highly related to mouse Ndr3 protein. Northern blot reveals that NDRG3 is highly expressed in testis, prostate and ovary. By in situ hybridization, the NDRG3 mRNA was localized to the outer layers of seminiferous epithelium, indicating that it may play a role in spermatogenesis.     

Mapping and Expression Analysis of Chicken NDRG1 and NDRG3 Genes

N-myc downstream-regulated genes 1 and 3 (NDRG1 and NDRG3) are members of the alpha/beta hydrolase superfamily. Phylogenetic analysis of the family demonstrated that human NDRG1 and 3 belong to a subfamily. The mapping and gene expression patterns of these genes represent one step toward further investigation into their possible roles in the chicken (Gallus gallus). To map these genes in the chicken chromosome, a 6000 rads chicken-hamster radiation hybrid panel (ChickRH6) was used. Primers were designed according to the published human sequences for amplification of those two genes. We compared the corresponding human mRNA sequences with the predicted coding sequences of the chicken NDRG1 and 3 genes and found that the assembled contigs shared a high percentage of similarity with the human genes. PCR of samples from ChickRH6 revealed that the locations of NDRG1 and 3 are linked to the markers MYC (58 cRs away, LOD score 4.52) and SEQ0265 (10 cRs away, LOD score 17.81), respectively. This result adds two new markers to the chicken RH map, and it reinforces that the RH technique is indeed a powerful tool for mapping genes due to its rapidity, precision, convenience, and reproducibility. In addition, we detected the gene expression and distribution of chicken NDRG1 and 3 in seven tissues, including heart, liver, spleen, lung, muscle, brain, and thymus, by RT-PCR, and found that NDRG1 is relatively ubiquitously expressed in all the tested tissues and highly expressed in heart and liver, whereas NDRG3 is high in heart, muscle, and brain.