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Review	Pdf file on Synergy OPEN omments
Acta Biochim Biophys Sin 2008, 40: 625-635
doi:10.1111/j.1745-7270.2008.00434.x

NDRG2: a Myc-repressed gene involved in cancer and cell stress

Libo Yao*, Jian Zhang, and Xuewu Liu

The Institute of Molecular Biology and the State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xi抋n 710032, China

Received: May 18, 2008�������

Accepted: May 30, 2008

This work was supported by grants from the Changjiang Scholars and Innovative Research Team in Universities of China (No. PCSIRT0459) and the National Natural Science Foundation of China (Nos. 30228012, 30370315, 30570676, 30670452, 30700416 and 06G092)

*Corresponding author: Tel, 86-29-84774513; Fax, 86-29-84774513; E-mail, [email protected]

As a master switch for cell proliferation and differentiation, Myc exerts its biological functions mainly through transcriptional regulation of its target genes, which are involved in cells interaction and communication with their external environment. The N-myc downstream-regulated gene (NDRG) family is composed of NDRG1, NDRG2, NDRG3 and NDRG4, which are important in cell proliferation and differentiation. This review summarizes the recent studies on the structure, tissue distribution and functions of NDRG2 that try to show its significance in studying cancer and its therapeutic potential.

Keywords�� ��N-myc downstream-regulated gene; tumor suppressor; cell stress

Myc was among the earliest oncogenes identified and has been the subject of intensive study in recent years. There has been a strong driving force to explore the function and regulation of Myc in cell proliferation, differentiation and apoptosis as well as its role as an important transcriptional factor [1].

As a master switch for cell proliferation and differentiation, Myc exerts its biological functions mainly by transcriptional regulation of its target genes [2]. The identification of authentic Myc target genes, both direct and indirect, is important in understanding the mechanisms it uses to regulate cell behavior [3]. A significant fraction of Myc-repressed genes are involved in cells interaction and communication with their external environment. It is especially interesting to note that several of these targets have been shown to possess tumor suppressor and anti-metastatic properties [4].

N-myc downstream-regulated gene 2 (NDRG2) [5-7], together with NDRG1 [8-11], NDRG3 [6,12], and NDRG4 [13,14], constitute the NDRG gene family, a new class of Myc-repressed genes. Accumulated data have shown the importance of this gene family in cell proliferation and differentiation. Significant attention has been paid to the NDRG gene family due to its potential as a tumor suppressor as well as its involvement in other diseases. Kovacevic et al and Ellen et al reviewed NDRG1抯 structure, regulation of gene expression, and function in normal and disease states [15,16]. This review focuses primarily on NDRG2, a member of the NDRG gene family that, like NDRG1, has been significant in cancer studies and has shown therapeutic potential. Comparisons of the structure and function of different members of the NDRG family are also discussed.

Gene and Protein Structure of NDRG2

Shimono et al first used 揘DRG to describe a gene that was identified as representative of those that expressed higher N-myc in the knockout mouse embryos [11]. The NDR1 (murine NDRG1) gene was augmented 20-fold in the mutant embryos at 10.5 d post coitus, which is indicative of repression by N-myc. A negative correlation is shown between the expression of N-myc and NDRG1 in various developing tissues of the wild-type embryos. The same group identified NDRG2 and NDRG3, which encode proteins highly related to NDRG1. However, NDRG2 and NDRG3 are under spatio-temporal regulations that differ from NDRG1 [6], which are not activated in N-Myc mutants.

Human NDRG1 gene was actually discovered earlier than the mouse NDRG family, but it was given a different name and lacked recognition as a downstream-regulated gene of Myc protein. In 1997, Kokami et al discovered a gene in which expression was induced by reducing agent and tunicamycin in human umbilical vein endothelial cells [8]. The gene was termed as RTP, standing for reducing agent and tunicamycin responsive protein. Van Belzen et al cloned this same gene and called it DRG1 (differentiation related gene) [9]. They found that DRG1 expression decreased in colon adenoma and adenocarcinomas, but it increased 20-fold when colon cancer cell differentiation was induced.

Deng et al first described the human NDRG2 sequence as a protein containing an acyl-carrier protein (ACP)-like domain [17]. The gene was cloned by polymerase chain reaction-based subtractive hybridization from glioblastoma, using normal brain tissues as control. Other members of the human NDRG gene family were reported later (table 1). The human NDRG family consists of four members: NDRG1, NDRG2, NDRG3 and NDRG4 [5-14]. Some alternative splicing isoforms exist in NDRG2 and NDRG4 [13,18,19].

Members of the NDRG family exist in many species, including zebrafish [20], Xenopus laevis [21], Drosophila melanogaster, Caenorhabditis elegant and Dictyostelium discoideum as well as in plants, such as sunflowers [22]. However, there is no protein with a significant similar sequence to NDGR2 in procaryotes, fungi and protozoa, which means that the distribution of NDRG family members is restricted to metazoa and plants.

Although NDRG members do not possess a clear functional peptide motif, they do share some well conserved residues. The percentage of shared residue identities among members is approximately 60% (Fig. 1) [13]. Phylogenetic analysis revealed that NDRG1 and NDRG3 belong to one subfamily, whereas NDRG2 and NDRG4 belong to another [7].

The Joint Center for Structural Genomics presented the partial crystal structure (residue 40-313) of mouse NDRG2 to the RCSB Protein Data Bank (2QMQ) (http://www.pdb.org/pdb) [23]. It contains 15 helices and 10 strands. Two domains can be recognized in the construct, an a/b hydrolase catalytic domain and a cap domain. Although the structure analysis grouped NDRG2 protein into a/b hydrolase superfamily, the key motif of catalytic activity in hydrolase seems absent. The catalytic triad in a specific order (nucleophile-acid-histidine), such as Ser-His-Asp/Glu triad motif, which exists in other hydrolase members [24], is not found in the structure of NDRG2 using multiple sequence alignment.

NDRG2 contains several potential phosphorylation sites and has been confirmed to be phosphorylated in certain cells by different protein kinases [25-27]. NDRG2 phosphorylation was increased in C2C12 cells (mouse myoblast cell line) co-overexpressing either PKC or Akt and stimulating insulin in a wortmannin- and palmitate-inhibitable manner, supporting a direct role for Akt. Thr348 is the major phosphorylation site for Akt, whereas PKC phosphorylates Ser332 [25]. NDRG2 phosphorylation was also observed in rabbit skeletal muscle extracts. It was phosphorylated rapidly by serum and glucocorticoidinduced kinase 1 (SGK1), but not by Akt-a. SGK1 phosphorylated NDRG2 at Thr330, Ser332 and Thr348 in vitro [26]. All three residues were phosphorylated in skeletal muscle from wild-type mice, but not from mice that do not express SGK1. NDRG1 was also phosphorylated by SGK1 at Thr328, Ser330 and Thr346 (equivalent to NDRG2抯 Thr330, Ser332 and Thr348) as well as at Thr356 and Thr366. Interestingly, Thr356 and Thr366 are located within identical decapeptide sequences, GTRSRSHTSE, which is repeated three times in NDRG1 but absent in other members of the NDRG family. These threonines were phosphorylated in NDRG1 in the liver, lung, spleen and skeletal muscle of wild-type mice, but not in SGK1-/- mice. The phosphorylation of NDRG1 by SGK1 transformed it into an excellent substrate for glycogen synthase kinase 3, which could then phosphorylate the Ser342, Ser352 and Ser362 of NDRG1 in the repeat region [26]. Furthermore, the phosphorylation of NDRG2 at T330 and T334 were detected by MALDI-TOF/TOF and nano-LC-ESI-MS/MS analysis of hippocampus protein extract from rats [27].

Tissue Distribution of Ndrg2

The expression pattern of human NDRG2 gene in various cells and tissues were first analyzed at the mRNA level [5,7]. Northern blot analysis using a human RNA master blot revealed that NDRG2 exists as a 2.0 kb single transcript. Prominent hybridization signals were detected in the muscle, brain, heart, liver and, to a lesser extent, in the kidney. In adult tissues, the highest expression level was found in the salivary glands, various neural tissues and skeletal muscles [5]. Furthermore, NDRG2 mRNA is nearly undetectable in the thymus, bone marrow, testis and peripheral blood leukocyte. This expression pattern suggests an inverse correlation between the level of NDRG2 gene expression and the rate of cell proliferation. In addition, the NDRG2 transcript was not detected in any of the tumor cell lines examined. These cell lines included leukemia (HL-60, K-562, and MOLT-4), lymphoma (Burkitt抯 lymphoma I, Raju and Daudi), lung carcinoma (A549), and colorectal carcinoma (SW480) [5].

Regarding the cellular and tissue distribution of NDRG proteins, human NDRG1 protein was first reported to be found mostly in epithelial cells [28]. Later, Wakisaka et al described rat NDRG1 protein expression in the kidney and brain [29]. The localization of NDRG1 protein in the kidney changed from the proximal convoluted tubules to the collecting ducts between 10 d and 20 d postnatal. In the brain, a change in cellular expression was also found from the hippocampal pyramidal neurons to the astrocytes in the gray matter during the same postnatal period.

Hu et al used an anti-NDRG2 monoclonal antibody to analyze the expression pattern of NDRG2 protein in mouse embryos at various gestational ages and in a variety of adult mouse tissues [30]. NDRG2 immunostaining 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 generally lower in the early stages of development and markedly increased during later stages. NDRG2 protein was also observed in a variety of adult mouse tissues, particularly in the heart and brain [30].

NDRG1 and NDRG2 protein expression has remarkable differences. NDRG1 protein commonly exists in various epithelia, including glandular epithelium [28], whereas NDRG2 is not detected in most epithelia, including glandular epithelium [30]. In terms of organ distribution, a differential expression between NDRG1 and NDRG2 has been noted in the heart, brain, testicles, ovaries and uterus. For instance, NDRG2 protein is highly positive throughout cardiac muscle [30], whereas NDRG1 protein is not expressed at all despite the existence of its mRNA [28]. NDRG2 is distributed widely in the brain, especially at high levels in the midbrain, cerebellum, medulla and thalamus [31], whereas NDRG1 is mainly found in the cortex and hippocampus [29].

Okuda et al recently made a precise comparison of the distribution of the NDRG family of proteins in the central nervous system [32]. They demonstrated that NDRG1 and NDRG2 were localized in the oligodendrocytes and the astrocytes, respectively, in the cerebrum. In the cerebellum, NDRG1 and NDRG4 were localized in Purkinje cells, while NDRG2 was in Bergmann glial cells. Shen et al further confirmed the NRDG2 protein抯 localization in the astrocytes by glial fibrillary acidic protein co-staining [31]. Interestingly, NDRG2 expression increased while the glioma cells were differentiating into astrocytes. These expression patterns revealed the cell type-specific and ubiquitous localization of the NDRG family of proteins. Each NDRG member may play partially redundant roles in specific cells in the brain [32].

In most of the reports, NDRG2 protein exists primarily in the cytoplasm [5,7,28,31,32], but it is also associated with the cell membrane and adherens junctions [28], even in the nuclei [30,31]. The nuclear staining of NDRG2 was initially observed in some cells in the midbrain of mice [30], and then confirmed in cells with astrocyte-like morphology in the hippocampus and cerebral cortex as well as in the olfactory bulb tissue [31]. Okuda et al found that NDRG3 was detected in the nuclei in most cells [32]. More importantly, NDRG1 and NDRG2 were translocated into nuclei upon cell stress [34,35]. The NDRG1-heat shock cognate protein 70 complex also transiently appeared in the nuclear fraction of activated mast cells [35].

The NDRG members have different tissue expression patterns, indicating that they may play distinct roles. More distribution information of NDRG proteins, especially in human tissues, is needed to better understand their function. The mechanisms and the conditions of nuclear localization of NDRG1 and NDRG2 proteins should be investigated more extensively.

NDRG2 alterations in human cancer

The various NDRG family members are reportedly intimately involved in cellular differentiation and development. NDRG1 has been associated with differentiation [9], embryo development [11], and tumor suppression [36,37]. Reduced expression of NDRG1 has been implicated in cancer cell proliferation and metastasis [15,16,34,36-40].

Given the fact that NDRG2 was cloned as a down-regulated gene in glioblastoma [17], several research groups investigated and confirmed its differential expression between tumor and normal tissues [5,41-48]. Deng et al analyzed NDRG2 mRNA levels in six normal brain tissues, 27 cases of human glioblastoma, 13 cases of low-grade glioma (from grade I to grade III), and six human high-grade astrocytoma/glioblastoma cell lines [5]. Their results demonstrated that the expression of NDRG2 was significantly reduced in 56% of human glioblastoma tissue samples and 100% of cell lines, as compared to expression in the normal brain or the low-grade glioma samples.

Lusis et al found that NDRG2 expression was consistently down-regulated in grade III meningioma at both the transcript and protein levels in independent sets of clinically and pathologically diverse cases [41]. Loss of NDRG2 expression was also seen in a subset of lower-grade meningiomas, including atypical meningiomas (WHO grade II) with clinically aggressive behavior. In the Norwegian cohort, mRNA levels of NDRG2 were significantly reduced in colorectal carcinoma when compared with those in the healthy controls. There was a trend for a decrease in NDRG2 levels with increasing Dukes stage [42]. Choi et al demonstrated that only two gastric cancer cell lines, SNU-16 and SNU-620, expressed NDRG2 among seven gastric cancer and two non-cancer cell lines [43]. NDRG2 was highly expressed in normal gastric tissues, but gastric cancer patients were divided into NDRG2-positive and NDRG2-negative groups. The survival rate of NDRG2-negative patients was lower than that of NDRG2-positive patients. It was confirmed that the loss of NDRG2 expression was a significant and independent prognostic indicator in gastric carcinomas by multivariate analysis [43].

Other groups also reported decreased NDRG2 gene expression in different cancer tissues, such as breast cancer [44], liver cancer [45], gastric cancer [46], oligodendroglial tumours [47], and skin cancer [48].

Hypermethylation is one of the most important attributes in the down-regulation of the NDRG2 gene. The loss of NDRG2 expression was significantly associated with hypermethylation of the NDRG2 promoter [41] in meningioma and several breast cancer cell lines [44]. In addition, the heterozygous deletion of NDRG2 was confirmed in breast cancer cell line MCF-7. It was also noticed that mutation [-13 bp (C>T)] of the NDRG2 core promoter significantly reduced NDRG2 activity in vitro [44].

While there have yet to be any controversial results reported about NDRG2 reduction in a variety of cancers, the same is not true of NDRG1. Though most research has reported down-regulation of NDRG1 expression in colon, breast and prostate cancers, some studies have reported increased expression of NDRG1 protein in colon and prostate malignancy [49,50]. The explanation for the inconsistent results is that NDRG1 expression patterns may reflect the prostatic epithelium抯 varying responses to hypoxia and androgens in African-American and Caucasian patients [50].

Collectively, these data identify NDRG2 as the candidate tumor suppressor gene and suggest that NDRG2 may be a useful and functionally relevant biomarker for predicting aggressive forms of cancer.

A recombinant adenovirus designed to preferentially eliminate p53-negative cells has shown that the loss of tumor suppressor activity may be therapeutically exploited [51]. Based on NDRG2抯 down-regulation in cancer tissue, several studies have followed it with interest to determine its potential as a target for cancer treatment. Most of these studies reported on the inhibitory effect of NDRG2 overexpression on tumor malignancy. The transfection of human glioblastoma U373 and U138 cells with a complementary DNA encoding NDRG2 was shown to markedly reduced cell proliferation [5]. NDRG2-silenced SNU-620 (gastric cancer) cells exhibited slightly increased proliferation and cisplatin resistance [43]. Additionally, inhibition of NDRG2 decreased Fas expression and Fas-mediated cell death. The inhibition of cell proliferation by NDRG2 overexpression in malignant cancer cells was also reported in other cancers, such as liver [52], lung [34], and breast cancers [44]. It was reported that NDRG2 overexpression in malignant breast cancer cells specifically inhibits Akt phosphorylation and induces phosphorylation of p38 mitogen-activated protein kinase and SAPK/JNK. Meanwhile, JAK2 or STAT3 activation in both resting and IGF-stimulating cells was inhibited by NDRG2 expression, implicating NDRG2 as a growth inhibitory gene in signal transduction pathways of breast tumor cells [53].

Considering the similar inhibitory effect of NDRG2 and NDRG1 on tumor cells, it is worth testing these targets for cancer treatment using gene manipulation strategies as well as small chemical treatments.

Control of NDRG2 gene Expression by Myc and other factors

Bioinformatics analysis of NDRG2 revealed several binding sequences for different transcription factors [54], which are mostly involved in growth regulation and early differentiation of cells. Some of those factors, such as Wilms tumor gene 1 (WT1) protein, hypoxia-induce factor-1a (HIF-1a) and glucocorticoids, up-regulate NDRG2 expression [34,55,56]. HIF-1a also up-regulates NDRG1 expression, whereas WT1 protein and glucocorticoids only regulate NDRG2 expression.

WT1 protein is a transcriptional regulator that is highly expressed in immature hematopoietic progenitor cells and in the majority of patients with acute and chronic myeloid leukemia. A WT1 binding site exists upstream of the NDRG2 promoter (-379 to -391 bp). Svensson et al found that WT1 indirectly or directly induced the expression of NDRG2 mRNA in CD34+ cells and in leukemic U937 cells through an oligonucleotide array approach [55]. Morevoer, a novel starting site for NDRG2 expression appeared to be used in WT1-transduced cells only, suggesting that this promoter is utilized preferentially when high levels of WT1 are present [55].

Sequence analysis of the human NDRG2 gene promoter revealed three putative hypoxia-responsive elements (HRE) motifs: HRE-1 from -188 to -183 bp; HRE-2 from -371 to -367 bp; and HRE-3 from -377 to -373 bp. Wang et al provided evidence that the expression of NDRG2 was regulated by HIF-1a in tumor cells upon hypoxia, and HRE1 could directly bind HIF-1a in vivo [34]. They suggested that HRE-1, HRE-2 and HRE-3 in the NDRG2 promoter are closely related to the regulation of HIF-1a. HRE-1 is more important than HRE-2 and HRE-3 because the deletion or mutation of HRE-1 in the NDRG2 promoter resulted in a dramatic decrease of luciferase activity induced by HIF-1a, whereas the deletion or mutation of HRE-2 and HRE-3 only resulted in a slight decrease of that activity.

Glucocorticoids reportedly up-regulate NDRG2 gene expression in rat brains, though a specific binding site for glucocorticoids receptor has not been identified [56].

More detailed studies have been done about the up-regulation of NDRG1 gene expression than NDRG2. NDRG1 is controlled by several known cell differentiation reagents [38], such as ligands [e.g. peroxisome proliferator-activated receptor gamma (troglitazone and BRL46593) and retinoid X receptor (LG268)] and histone deacetylase inhibitors (e.g. trichostatin A, suberoylanilide hydroxamic acid). The expression of NDRG1 was induced by DNA-damaging agents as well as by enforced expression of wild-type p53 [57,58]. The elevation of NDRG1 expression in G1 and G2/M phases was believed to have resulted from p53-mediated transcription activation, which is known to cause cell cycle arrest upon DNA damage. In addition to p53, other transcription factors, such as HIF-1, c-Jun/AP-1, E2a-Pbx1 fusion protein [59], were also implicated in the regulation of NDRG1 gene expression. HIF-1 is required for Ni2+ compound-induced NDRG1 expression. C-Jun/AP-1 plays an important role in Ca2+ ionophore and iron chelator [60], as well as in hypoxia-induced NDRG1 expression [39,61]. In addition, comparison of the noncoding sequence of the rat NDRG4 gene [19] and human NDRG2 gene with those of the orthologous mouse and human genes suggests that the AP-1 binding site is a candidate regulatory element.

As a downstream gene of Myc, mouse NDRG1 expression was found to be repressed by N-Myc and c-Myc [11]. The NDRG1 promoter activity was down-regulated by N-myc, and more strongly by the combination of N-myc and Max in the cotransfection assay [11]. This repressive effect was through the promoter region within 52 base pairs from the transcription start site. The effect of N-myc:Max was sensitive to trichostatin A, indicating the involvement of histone deacetylase activity in repressing the NDRG1 promoter [11]. In contrast, N-myc does not seem to regulate mouse NDRG2, since the expression of NDRG2 was not up-regulated in tissues of N-myc knockout mice [6].

The evidence that human NDRG1 is repressed by N-Myc overexpression in neuroblastoma cell lines suggests its similarity with mouse NDRG1 with regard to gene expression regulation. Human NDRG1 is strongly repressed in all tested neuroblastoma cell lines bearing N-myc amplification, as well as in a neuroepithelioma line with amplified c-MYC [62]. In vitro interaction of Myc protein with the NDRG1 core promoter was found in cells. The re-expression of NDRG1 in high-MYC neuroblastoma cells resulted in smaller cells with reduced colony size in soft-agar assays, further underscoring the functional significance of NDRG1 in human cancer cells [62].

Although N-myc does not seem to regulate mouse NDRG2, an elevated c-MYC mRNA level was found in human glioblastoma with reduced NDRG2 mRNA [5]. It suggests that human NDRG2 might be regulated differently than mouse NDRG2 as the target of Myc. Zhang et al provided evidence that the expression of human NDRG2 is down-regulated by Myc via transcriptional repression [63]. The ectopic expression of c-Myc dramatically reduces the cellular NDRG2 protein and mRNA levels. Furthermore, this confirmed the core promoter region of NDRG2 necessary for Myc repression on NDRG2 transcription and verified the interaction of Myc with the core promoter region both in vitro and in vivo. Moreover, the c-Myc-mediated repression of NDRG2 requires association with Miz-1 and possibly the recruitment of other epigenetic factors, such as HDACs, to the promoter [63]. Till now, Zhang's work concerning the transcriptional repression of NDRG2 by Myc is the relative explicit report about the mechanism research of NDRG family.

Compared with the sequences predicted to be regulated by various transcription factors in the promoter region of the NDRG gene family, only a few elements have been studied at present. A systematic analysis of these transcription factors is required to illustrate the gene regulation map of the NDRG family.

Ndrg2 Functions as a Cell Stress Responsor

It is well known that a number of cell stress conditions induce the expression of human NDRG1. The first clue about NDRG1's reaction to cell stress conditions was that homocysteine, the sulfhydryl group-containing amino acid, up-regulates the expression of this gene [8], which has also been confirmed by a number of subsequent studies [10,57,64-67]. For instance, DNA-damaging agents induced NDRG1 expression in a p53-dependent manner. NDRG1 protein demonstrated a cytoplasmic localization pattern with redistribution into the nucleus upon DNA damage [10]. Stein et al demonstrated that NDRG1 acts as an indispensable contributor during p53-induced caspase activation and apoptosis [57]. The hypoxia and nickel reagent could induce human NDRG1 expression in an HIF-1-dependent manner [65-67].

It is reasonable to predict the possibility that NDRG2 works as a cell stress responding molecule as well. Wang et al investigated the involvement of NDRG2 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 levels [34]. The expression of NDRG2 was regulated by HIF-1 in tumor cells under hypoxia, and HIF-1 directly bound to HREs in the NDRG2 promoter in vivo. Importantly, silencing or enforcing the expression of NDRG2 may strongly inhibit or increase apoptosis.

Similar to NDRG1 protein upon DNA damage, NDRG2 can be translocated from the cytoplasm to the nucleus [34,68]. However, there is no explicit nuclear localization signal (NLS) sequence identified in NDRG2 protein. Although a NLS is the most common type of nuclear import element, other sequences are important for targeting certain proteins to the nucleus. For example, adenomatous polyposis coli and breast cancer 1 were recently shown to enter the nucleus via pathways that are independent of their NLS [69]. It could be speculated that NDRG2 may have its own motif that is directly or indirectly responsible for its nuclear translocation. Wang et al confirmed that the segment (101 to 178 amino acids) of NDRG2 is responsible for its nuclear translocation [34].

Although the exact physiological significance of Ndrg2 in cell stress is unknown, the role of NDRG2 and NDRG1 in controlling cell cycle progress and apoptosis signal sensitivity has been well established [34,38,57]. Considering the phenomenon that many tumor suppressors belong to cell stress responding genes, it would be interesting to explore the detailed working mechanism of Ndrg proteins under stress. It may provide more clues of Ndrg protein as a therapeutic target of cancer.

Ndrg2's Function in Nervous System

The NDRG gene family is expressed widely in the nervous system. Each member has its own distribution priority in different parts of the brain and cell-type specificity [32], suggesting this gene family may have a complicated role in nervous system. Disease-related gene structure or expression changes were noticed both in NDRG1 and NDRG2 [70-72]. Hereditary motor and sensory neuropathy-Lom (HMSNL), a severe autosomal recessive form of Charcot-Marie-Tooth disease, is a common cause of disability in adulthood. Kalaydjieva et al identified NDRG1 as the causing gene of HMSNL [70]. A single point mutation, a premature-termination codon at position 148 was confirmed in HMSNL patients. Mutations in NDRG1 accounted for 2.88% of the overall group of patients investigated and for 4.68% of the cases with demyelinating neuropathies [71]. HMSNL is a feature of Schwann cell dysfunction and concomitant early axonal involvement. The demyelination of peripheral nerves is one of the major pathologic changes in HMSNL patients, implicating NDRG1 in Schwann cell signaling. The NDRG1 deficiency mice model provided more evidences supporting NDRG1抯 important function in Schwann cells differentiation [72]. Progressive demyelination in peripheral nerves is the major phenotype in NDRG1 knockout mice, suggesting that this protein is essential for myelin sheath maintenance. Hirata et al observed NDRG1 expression during the course of Wallerian degeneration and ensuing regeneration after injuring mouse sciatic nerves [73]. They found that NDRG1 expression was maintained in the early stage of myelin degradation but markedly reduced at the end stage. Intriguingly, NDRG1 expression increased at the stage of remyelination, with immunoreactivity stronger than that in intact nerves [73].

Because brain tissue is one of the most abundant tissues expressing NDRG2 [5,7,55], the important function of this protein in the nervous system was undoubtedly expected and was confirmed by several studies [55,74,75]. Mitchelmore et al found that NDRG2 is up-regulated at both the RNA and protein levels in the brains of patients with Alzheimer抯 disease [74]. Expression of NDRG2 in affected brains was found in cortical pyramidal neurons, senile plaques and cellular processes of dystrophic neurons. Overexpression of two splice variants encoding a long and short NDRG2 isoform in hippocampal pyramidal neurons of transgenic mice resulted in localization of both isoforms to dendritic processes. Takahashi et al demonstrated that chronic treatment with a tricyclic antidepressant (i.e. imipramine) and a selective serotonin reuptake inhibitor (i.e. sertraline) reduced the expression of NDRG2 mRNA and protein in the rat frontal cortex [75]. Repeated electroconvulsive treatment also significantly decreased NDRG2 expression in this region of the brain, implying that NDRG2 may be associated with treatment-induced adaptive neural plasticity in the brain, a chronic target of antidepressant action [75]. As antidepressants may alleviate symptoms of depression by reversing the effects of glucocorticoids, increased NDRG2 mRNA resulting from glucocorticoid treatment in astrocytes suggests that further study of NDRG2 regulation and function in glia could contribute to a better understanding of the pathogenesis and treatment of depression [56].

In addition, other studies reported the effects of NDRG2 and NDRG4 on neural cell differentiation [76,14]. NDRG2 promotes neurite outgrowth of NGF-differentiated PC12 cells [76]. The cells having decreased levels of the NDRG4 protein (antisense construct of rat NDRG4 complementary DNA transfectants) extended shorter neurites than control cells in response to NGF or dibutyryl cAMP [14]. NGF-mediated activation of the transcription factor AP-1 was suppressed in the NDRG4 protein-diminished clones as compared with those in the control cells. NGF-induced phosphorylation of MEK and ERK was enhanced by NDRG4 protein [77]. Interestingly, NDRG4 mRNA increased during hot water epilepsy in a rat model [78].

Taken together, the NDRG family is a group of proteins expressed widely in brain. Some family members are expressed differentially in diseases of the nervous system and regulated by the agents targeting neurons or astrocytes. The study of the physiological and pathological roles of these family members in the nervous system will be helpful for understanding the mechanism of related diseases.

The Other Function of NDRG2

NDRG2 has also been reported to play roles in other functions, such as insulin action [25], aldosterone-mediated epithelial sodium channel (ENaC) function [79,18], and dendritic cell (DC) differentiation [80,81].

NDRG2 protein is probably involved in insulin action [25]. It was directly phosphorylated by endogenous Akt upon stimulation of muscle cells with insulin. The Akt-mediated phosphorylation of NDRG2 at Thr348 is inhibited by PKCq, which phosphorylated Ser332 of NDRG2. This crosstalk might represent one mechanism by which lipid-activated PKC interfere with insulin action [25].

Simultaneously, NDRG2 has been identified as an early aldosterone-induced gene in rat kidneys [18]. Recently, Wielp�tz et al found that NDRG2 may affect ENaC function in Xenopus laevis oocytes and rat thyroid cells [79]. Co-expression of NDRG2 significantly increased whole cell current in some, but not all, batches of oocytes tested. An NDRG2-induced increase in ENaC currents was accompanied by a similar increase in channel surface expression.

NDRG2抯 mRNA is nearly undetectable in the thymus, bone marrow, testes and peripheral blood leukocyte [5]. However, it was expressed in DCs derived from CD34+ progenitor cells and differentially regulated by maturation-inducing stimuli. The inhibition of DC differentiation by dexamethasone or vitamin D treatment decreased the expression of the NDRG2 gene in DCs. In addition, gene expression was induced in a myelomonocytic leukemia cell line, which is capable of differentiating into DCs in cytokine-conditioned culture [80]. The expression of activated leukocyte cell adhesion molecule is down-regulated specifically in DC differentiated from NDRG2 short interfering RNA-transfected monocytes. Furthermore, DCs differentiated from NDRG2 short interfering RNA-transfected monocytes showed a reduced ability to induce T-cell proliferation [81]. It was also reported that the expression of NDRG2 mRNA was induced by WT1 in CD34+ cells and leukemic U937 cells [54]. Therefore, NDRG2 is a cell differentiation regulator in some type of hematopoietic progenitors. Similar to NDRG2, NDRG1 may be a mast cell maturation-associated inducible protein [82,83].

Concluding remark

The studies on the function and regulation of the NDRG family have provided plenty of information indicating the multiple roles of this gene family. The most interesting findings about NDRG2 are as follows: (1) its expression negatively correlates to cancer progression and positively correlates to cell differentiation; (2) it is involved in cell stress response; (3) its expression changes in nervous system diseases; and (4) it may be an ENaC regulator. Future studies will need to evaluate NDRG2's potential as a biological marker or a therapeutic target of cancer. To elucidate the exact function and regulation mechanism of NDRG2, research of other NDRG family members will provide more information on the network controlled by Myc under normal and disease conditions.

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