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Acta Biochim Biophys |
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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, Wielpütz 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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