Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
In recent years, significant progress has been made
towards the understanding of molecular mechanisms that
control the growth of neuronal processes during the
development and maturation of the central nervous system (CNS).
Special attention has been paid to the extracellular matrix
molecules (ECM), which not only form as a physical
framework in the CNS, but also exert profound effects on cell shape
and behavior, including cell adhesion, spreading, migration,
proliferation, and differentiation[1]. In the brain, chondroitin
sulfate proteoglycans (CSPG) are prominent components of
the ECM and are assumed to play a particularly important role
in controlling neuronal differentiation and
development[2,3]. The mammalian brain contains many different types of CSPG
core proteins, and during development, these proteoglycans
exhibit a complex pattern of expression. As the CNS
deve-lops from early gestation into late postnatal ages, the amount
of CSPG steadily increases and reaches adult levels several
weeks after birth[4].
Versican is one of the major extracellular proteoglycans in
the developing and adult brains. It was originally isolated
from human fibroblasts and developing chicken limb
buds[5,6] and was also detected in normal human CNS and brain
tumors[7]. In the rat CNS, it was highly expressed in white
matter tracts and closely linked to
myelination[8]. The highly interactive nature of versican provides a basis for its
importance as a structural molecule to create loose and hydrated
matrices during the key events in development and disease.
By interacting either directly with cells or indirectly with
molecules that associate with cells, it regulates cell survival,
proliferation, adhesion, migration, and differentiation, as well
as ECM assembly and cell
phenotype[9_13].
Structurally, versican is made up of an N-terminal G1
domain, a chondroitin sulfate (CS) attachment region, and a
C-terminal G3 domain. The latter contains 2 epidermal growth
factor (EGF)-like repeats, a lectin-like motif, and a
complement binding protein-like
motif[14]. The CS sequence can be divided into 2 alternatively spliced domains, termed
CSα and CSβ. The alternative splicing of the versican gene generates
4 isoforms that are expressed in distinct spatiotemporal
patterns[6,10], namely V0, V1, V2, and
V3[14,15]. V0 contains both CSα and
CSβ; V1 and V2 possess only CSβ and CSα, respectively; and V3 contains neither
CSα nor CSβ. Versican is known to associate with a number of other molecules in
the ECM, including hyaluronan, tenascin, fibulin-1, fibrillin,
fibronectin, CD44, selectins, and the link
protein[16_21]. Studies of brain development and maturation have shown that
versican V1/V0 and V2 exhibit complementary expression
patterns in the CNS[22]. Although versican V1/V0 isoforms
are mainly expressed in the late stages of embryonic
development, they frequently associate with little adhesive,
fast-proliferating tissues that display a high ECM turnover
rate[23]. Versican V2, on the other hand, is mainly expressed
in adults and becomes a major CSPG in the mature
brain[24]. The different expression patterns of versican isoforms
suggest that they may play distinct roles in neuronal
differentiation and neurite outgrowth. Versican V2 inhibits neural
differentiation and neurite growth[25], whereas versican V1 induces
neural differentiation and promotes neurite
outgrowth[26]. These distinct functions of versican isoforms may be a
result of the different CS domains they possess.
Remodeling of the ECM occurs continuously as a part of
physiological processes, such as development, growth, and
aging. It also occurs in pathological processes, such as
traumatic and inflammatory lesions. The variation in the
relative composition of different proteoglycans thus
influences the organization and thereby the properties of the
matrix. Factors involved in the regulation of ECM
remodeling include cytokines, such as transforming growth
factor-β (TGF-β), which are known to be potent regulators in the
inflammatory process. Other cytokines, such as
pro-inflammatory cytokines interleukin-1β (IL-1β) and
TNF-α may also play important roles. They can alter the synthesis of
ECM molecules and have varying effects depending on the type
of cells involved. It has been well known that after injury to
the adult CNS, numerous cytokines and growth factors are
released that contribute to reactive gliosis and ECM
production and therefore limit the capacity for axon regeneration.
Previous reports also revealed that versican was upregulated
in the injured CNS and presented in the environment in which
axon regeneration failed[27]. However, little is known
concerning the effect of injury or pro-inflammatory cytokines on
the expression of versican isoforms.
In the present study, we examined in
vitro the temporal expressional profiles of versican in neural precursor cells
(NPC) and their linage cells, especially the oligodendrocyte
(OL) lineage cells. We compared the expressional patterns
of versican isoforms V1/V0 and V2 at different
developmental stages from NPC to mature oligodendrocytes. We
examined the effects of 2 pro-inflammatory cytokines,
TNF-α and IFN-γ, on the expression of versican isoforms in NPC
in vitro. The objective of this study was to determine the expression
and regulation of versican isoforms in oligodendrogliogenesis and the pathological environment.
Materials and methods
Cell culture The isolation and cultivation of NPC
derived from embryonic rat neural precursor cells were
prepared according to the method described by Hu
et al[28]. Briefly, NPC were isolated from the embryonic spinal cords
(SC) of E16 Wistar rats. After removing the amnion and dura,
the SC were dissected in sterile dishes containing ice-cold
Leibovitz's L-15 medium (Invitrogen, Grand Island, NY,
USA), dissociated by gentle mechanical pipetting through
fire-polished Pasteur pipettes to achieve single-cell
suspen-sions, then filtered through a 70 mm nylon mesh. The
dissociated viable cells were then seeded into a T25 Corning tissue
culture flask (Corning, Corning, NY, USA) containing growth
medium, at a density of 1×105 cells/mL, then incubated in a
humidified atmosphere containing 5% CO2 at 37
°C. The culture medium for NPC was composed of 1×Dulbecco's
modified Eagle's medium/F12 (Invitrogen, USA), 1×N2
(Invitrogen, USA), 1% B27 (Invitrogen, USA), 0.06% glucose,
2 mmol/L glutamine (Invitrogen, USA), 1.34 mmol/L sodium
bicarbonate, 0.5 mmol/L HEPES
(N-2-hydroxyethylpipera-zine-N''-2-ethane sulfonic acid), 2
mg/mL heparin, freshly added 20 ng/mL EGF (all from Sigma, St Louis, MO, USA),
and 20 ng/mL Basic fibroblast growth factor (bFGF,
Invitrogen, USA). After incubation for 3 or 4 d, the cells
developed into visible neurospheres of 50_200 cells/sphere
and then were mechanically pipetted into single cells for
passage. The NPC of passage 2 were collected for RNA
extraction or immunocytochemistry (ICC).
For cytokine stimulation, the dissociated NPC of
passage 2 were incubated with different
concentrations of
recombinant rat IFN-γ or TNF-α or a combination (both from
PeproTech EC, London, UK) in NPC culture medium, 8 h
after seeding. The control groups were grown under
identical conditions except for the cytokines. Forty eight hours
later, the cells of both groups were harvested for RNA
extraction.
To induce the differentiation of NPC, the dissociated cells
in the single-cell suspension were seeded onto
poly-L-lysine (200 mg/mL, Sigma, USA) coated coverslips in 35
mm dishes at a density of 5×104 cells/coverslip. Then the
growth factors were removed and 1% fetal bovine serum
(FBS, Invitrogen, USA) was added. The cultures were
allowed to differentiate for 5_7 d in vitro before being fixed
for immunostaining.
All embryonic rats were obtained from female pregnant
Wistar rats bred in the Animal Care Facility at Shanghai
Jiaotong University School of Medicine (Shanghai, China).
All animal care was performed in accordance with the
National Institute of Health's Guide for the Care and Use of
Laboratory Animals.
Induction and passage of oligodendrocyte precursor cells
The induction of oligodendrocyte precursor cells (OPC) from
NPC was performed as previously
described[29,30] with modifications. Briefly, freshly dissociated NPC from SC were
seeded at 1×105 cells/mL in NPC medium supplemented with
10 ng/mL bFGF and EGF for 1 or 2 d to develop small
neurospheres and then gradually replaced with fresh OPC
medium every other day 3 times. The OPC medium
comprised of NPC medium (except bFGF/EGF) with the addition
of 0.1% bovine serum albumin (Amresco, Solon, OH, USA),
10 ng/mL biotin (Sigma, USA), 10 ng/mL recombinant rat
platelet-derived growth factor (PDGF-AA, R&D,
Minnea-polis, MN, USA), and 15 ng/mL bFGF (Invitrogen, USA).
During the induction process, the cells bearing the
characteristic morphology of OPC migrated from the neurospheres
and attached to the bottom of the flask. After removal of the
necrotic spheres and fragmented cells, the OPC were
cultured for an additional 5_7 d until visible oligospheres of
50_200 cells/sphere were formed.
For OPC passage, cultures were incubated with 1.2 mL
accutase solution (Innovative Cell Technologies, San Diego,
CA, USA) for 12 min at 37 °C. After the OPC/oligospheres
detached entirely from the bottom of the flask, the cell
suspension was harvested, gently triturated using a
fire-polished Pasteur pipette to dissociate spheres, and then
centrifuged at 134×g for 8 min at 20 °C. The dissociated cells
were resuspended and reseeded into a T25 flask containing
fresh OPC medium at a density of
1×105 cells/mL. After 7_10 d, new oligospheres formed, which were passaged
again. The OPC of passage 2 were harvested for later
experi-ments, such as the identification of OPC purity, RNA extract,
ICC, or induction of OPC differentiation. To examine the
purity of OPC, the cells were plated onto
poly-L-lysine (200 mg/mL, Sigma, USA) coated coverslips in 35 mm dishes and
cultured in fresh OPC medium for 3 d, then fixed for
immunostaining.
Differentiation of OPC To induce OPC
differentiation in vitro, the dissociated OPC of passage 2 were seeded
into a poly-L-lysine (100 mg/mL, Sigma, USA) coated T25
flask or coverslips at a density of
2×105 cells/mL or
5×104 cells/coverslip, respectively. The growth medium consisted
of OPC medium without PDGF-AA/bFGF, but with 1% FBS
(Invitrogen, USA) and 30 mmol/L thyroid hormone
(tri-iodothyronine [T3], Sigma, USA). The OPC were allowed to
differentiate for 4 d (4DIV) or 14 d (14DIV) in
vitro and then collected for RNA extraction or ICC analyses. Antibodies
against A2B5, O4, O1, and myelin basic protein (MBP),
respectively, were used to identify OPC and
oligodendrocyte lineage cells at different developmental stages.
ICC Immunofluorescence double-labeling was used for
the colocalization of versican and cell-specific markers. For
NPC, floating spheres were fixed in 4% paraformaldehyde
(PFA) overnight, washed in 0.01 mol/L phosphate-buffered
saline (PBS; pH 7.4), cryoprotected in PBS containing 30%
sucrose, embedded in Optimal Cutting Temperature (O.C.T)
Compound (Sakura FineTec, Torrance, CA, USA), and
sectioned with a cryostat. The cell cultures on
poly-L-lysine coated coverslips were fixed with 4% PFA for 10 min at room
temperature (RT), washed, and stored in 0.01 mol/L PBS (pH
7.4). The sections of neurospheres or cell cultures were
blocked in 10% goat serum in PBS (for cell surface staining)
or 0.3% Triton X-100-containing 10% goat serum in PBS (for
intracellular staining) for 1 h at RT and incubated with the
following primary antibodies overnight at 4 °C: rabbit
antiversican antibody (1:400, a kind gift from Prof Yi-ping
ZHANG, University of California, Irvine, CA, USA); the
monoclonal mouse antibodies against nestin (1:800, BD
Pharmingen, San Jose, CA, USA) for NPC, βIII-tubulin
(1:800, Sigma, USA) for the neurons, glial fibrillary acidic
protein (GFAP, 1:200, Sigma, USA) for the astrocytes, or Rip
(1:25, a gift from Dr Scott R WHITTEMORE, University of
Louisville, Louisville, KY, USA) for the oligodendrocytes;
the monoclonal mouse antibodies Immunoglobulin M (IgM)
against A2B5 (1:200), O4 (1:800), O1 (1:800, all from R&D,
USA), and the monoclonal mouse antibodies against MBP
(1:40, Oncogene Corp, Seattle, WS, USA) for identifying OPC
and/or OL. After washing with PBS, the sections and cell
cultures were incubated for 60 min at 37 °C with the
appropriate secondary antibodies: fluoresceinisothiocyanate
(FITC)-conjugated goat antirabbit Immunoglobulin G (IgG, 1:80,
Sigma, USA) for versican, rhodamine-conjugated goat
antimouse IgM (1:200, Santa Cruz Biotechnology, Santa Cruz,
CA, USA) for A2B5, O4 and O1, and rhodamine-conjugated
goat antimouse IgG (1:100, Sigma, USA) for the others. The
slides or coverslips were rinsed and mounted with Gel/Mount
aqueous mounting media containing Hoechst 33342 (1
mg/mL, Sigma, USA), a fluorescent nuclear dye. The images
were acquired using an Olympus BX60 microscope (Olympus,
Tokyo, Japan) equipped with a digital camera and SPOT
4.0.1(G) software (Diagnostic instruments, Sterling Heights, MI,
USA). In all the experiments, primary antibody omission
controls were used to confirm the specificity of ICC. The
double-immunostaining of CD4 (the specific marker for Th
lymphocyte) and versican for the sections of lymphnodes
were also applied to confirm the specificity of the antiversican
antibody. For NPC and OPC counting, at least 5
randomly-selected fields with more than 200 cells were counted, and
more than 300 cells for each individual was counted for the
others.
RNA extraction and single-stranded cDNA
synthesis Total cellular RNA was extracted using TRI reagent
(Molecular Research Center, Cincinnati, OH, USA)
according to the manufacturer's instructions. Residual DNA
contamination was eliminated by DNase I (Promega, Madison,
WI, USA) treatment (5 U DNase I for 45 min at 37 °C). The
integrity of the RNA samples was checked by the density
ratio of 28S against 18S RNA in 1.0% agarose gel
electro-phoresis. The concentration and purity of RNA was
determined by repeated spectrophotometric measurements at 260
and 280 nm. 1 mg RNA was subjected to synthesize
single-strand cDNA with the reverse transcription system from
Promega (USA). The 20 mL reaction mixture contained 15 U
AMV reverse transcriptase, 10 U recombinant
RNasin® ribonuclease inhibitor, 5 mmol/L
MgCl2, 1×reverse transcription buffer, 0.5
mg random primer, and 1.0 mmol/L dNTP mixture. After denaturation at 70 °C for 10 min, the reaction mixture
was incubated at 42 °C for 50 min followed by heat
inactivation of the enzyme at 95 °C for 5 min, then cooled on ice for 5
min and stored at -20°C.
Semiquantitative PCR Initial dilution and the cycle
series experiments were carried out to determine the linear
exponential amplification phase and select the appropriate
quantity of input cDNA and cycle numbers in PCR. The
housekeeping gene β-actin was used as the internal control.
The primer sequences were as follow: V1/V0: forward 5'-GAT
GTA ACA ACC ACT CCG TCA G-3', reverse 5'-CGC AAC ACT TTC ATA CAG GC-3'; V2: forward 5'-TCA AAG CCT
CCT GTA ATG C-3', reverse 5'-CCG ACA AGG GTT AGA GTG A-3'; and
β-actin: forward 5 -ATT GTA ACC AAC TGG GAC G-3', reverse 5'-TTG CCG ATA GTG ATG ACC T-3'.
The 25 mL PCR reaction contained 1.5 U
Taq DNA polymerase (Promega, USA), 1×PCR buffer, 1.0
mL cDNA,
1.5 mmol/L MgCl2, 0.2 mmol/L dNTP mix,
0.4 mmol/L primers for V1/V0 and V2, and 0.06
mmol/L primers for β-actin. The reactions were incubated for 5 min at 94 °C, followed by 25
cycles of denaturation (30 s at 94 °C), annealing (30 s at
57 °C), elongation (30 s at 72 °C), and final extension (10
min at 72 °C). Then 10 mL of PCR products was separated on
1.5% agarose gels with 0.1% ethidium bromide and
photographed under UV illumination. The ratio value of the signal
intensity of the tested genes versus β-actin was quantified
with the National Institute of Health Image.
Real-time PCR Real-time PCR was carried out on an
ABI7900 PCR detection system (Applied Biosystems,
Foster City, CA, USA) using the SYBR Green PCR Master Mix
(Applied Biosystems, USA). To normalize the gene
expres-sion, an endogenous reference HPRT (hypoxanthine
guanine phosphoribosyl transferase, housekeeping gene)
was performed for parallel amplification. The primer sequences
were: V1/V0: The same as those in semiquantitative
RT-PCR above; V2: forward 5'-TCA AAG CCT CCT GTA ATG C-3',
reverse 5'-ATA GCA GGT GCC TCC AT-3'; and HPRT: forward
5'-CTC ATG GAC TGA TTA TGG ACA GGA C-3', reverse
5'-GCA GGT CAG CAA AGA ACTT ATA GCC-3'.
Each PCR reaction (total volume of 10 mL) contained 1.0
mL cDNA, 5.0 mL of 2×SYBR Green PCR Master Mix, and 0.4
mmol/L of each primer. The reaction solutions were
incubated at 50 °C for 2 min, then at 95 °C for 10 min, followed by
40 cycles of denaturation at 95 °C for 15 s, and annealing at
60 °C for 1 min. The PCR cycle number at which the
fluorescent crosses a threshold (CT value) could be related to the
amount of initial templates. The relative expression level of
target mRNA was calculated by the normalized expression of
the target gene with respect to the HPRT gene. The
difference of target gene expression between the treatment group
and the control group was computed with the equation:
Folds=2-DDCT, where DDCT is the difference between
the DCT of the treatment group and the DCT of the control,
DCT is the difference between the CT of the target gene and the CT
of the HPRT within the same sample.
Statistical analysis Data were presented as mean±SEM
values. Statistical analysis was performed by SPSS 10.0
(SPSS, Chicago, IL, USA), and one-way ANOVA with
post-hoc Tukey LSD (Least Significant Difference) test was used
to determine statistical significance. A P-value of less than
0.05 was considered statistically significant.
Results
Expression of versican in NPC and their
lineages After the dissociated NPC were plated in N2/B27 growth medium
supplemented with EGF and bFGF, most of them proliferated
rapidly to form floating neurospheres. Immunostaining of
the sectioned neurospheres indicated that nearly all the cells
within the spheres expressed the intermediate filament
protein (nestin), a marker for NPC (Figure1B). After withdrawal
of EGF and bFGF and the addition of 1% FBS, the NPC
began to differentiate into a mixture of
GFAP+ astrocytes (Figure1E),
βIII-tubulin+ neurons (Figure1H), and
Rip+ oligodendrocytes (Figure1K).
The double-immunofluorescence staining showed that
versican was expressed in nestin+ NPC (Figure1C),
GFAP+ astrocytes (Figure1F),
βIII-tubulin+ neurons (Figure1I), and
Rip+ oligodendrocytes (Figure1L). As the cells tested were
fixed and permeabilized before the ICC assay, the staining
should be the result of intracellular immunoreactivity. In the
differentiated cells, the versican staining was observed in
both the cell bodies and their processes. However, we could
not observe the staining of versican in CD4+
lymphocytes in the lymph node, which had been reported to not express
versican, and this negative staining result verified the
specificity of the antiversican antibody used (Figure1P_1R). To
exclude the possible influence on the expression of versican
in NPC and their lineages due to differentiation
in vitro, the neurons isolated from E18 rat embryonic cerebral cortex and
astrocytes, mainly type-1 astrocytes obtained from newborn
rats, were also tested for versican expression by ICC. The
results showed that both cell types differentiated
in vivo expressed versican (data not shown). We also observed the
location of versican in the spinal cord in
vivo by double-immunostaining (neural cell specific markers and versican)
of the spinal cord sections and obtained the same results as
that from the cell cultures in vitro (data not shown).
Expression of versican on oligodendrocyte lineage cells
A previous report revealed that versican was mainly a
product of oligodendrocyte lineage
cells[27]. To study the expressional profile of versican at different developmental stages
of oligodendrocytes, an in vitro model was established,
which could mimic the environment of
oligodendrogliogenesis in vivo and generate
A2B5+ OPC, O4+ pre-oligo-dendrocytes,
O1+ immature oligodendrocytes, and
MBP+ mature oligodendrocytes over time.
The OPC generated from NPC in this study displayed
bipolar and tripolar morphology (Figure 2A_2C) and most of
them (97.33%±3.06%, n=3) were positive for A2B5 (Figure
2B), a marker of OPC, indicating that the NPC-induced OPC
were highly purified. Immunofluorescent double-staining
showed that versican was expressed on the surface of soma
and processes of OPC (Figure 2A) colocalizated with A2B5
(Figure 2C).
When OPC were cultured in differentiation medium with
1% FBS and T3, they progressively extended more processes
and expressed more mature markers, such as O4, O1, and
MBP, while A2B5 decreased quickly.
Double-immunofluorescence staining showed that versican was expressed in
O4+ pre-oligodendrocytes (Figure 2D_2F),
O1+ immature oligodendrocytes (Figure 2G_2I), and
MBP+ mature oligodendrocytes (Figure 2J_2L). Versican was expressed on both
soma and processes of these cells. As the staining was
carried out on the fixed and unpermeabilized cells, it
indicated that the epitopes of versican were localized on the
extracellular surface of the glial membrane.
Expression patterns of versican isoforms on NPC and
oligodendrocyte lineage cells As we were unable to obtain
antibodies to distinguish V1 from V2, semiquanti-tative
RT-PCR was performed to examine the transcription levels of
versican V1/V0 and V2 in NPC and oligodendrocyte lineage
cells, that is, OPC, OL (4DIV), and OL (14DIV). The
characteristics of OL at these selected differentiated stages were
identified by immunofluorescence based on their
differentiation markers (Figure 3). The results showed that
O4+ cells increased from 11.95%±2.88% at OPC to 43.34%±5.48% at
OL (14DIV). O1+ and MBP+ cells could hardly be detected at
OPC, but along the OPC maturation, O1+ and
MBP+ cells increased from 37.19%±1.68% and 11.43%±2.70% at OL
(4DIV) to 65.26%±3.25% and 25.76%±1.31% at OL (14DIV),
respectively, while A2B5+ cells dropped greatly from
97.33%±3.06% at OPC, to 20.99%±3.50% at OL (14 DIV).
Consistent with the results of immunostaining for versican
expression, the RT-PCR result revealed that the mRNA of
V1/V0 and V2 were present in NPC as well as oligodendrocyte
lineage cells, but the expression patterns of 2 isoforms at
different development stages were different (Figure 4). The
statistical analysis showed that the expression of V1/V0 in
NPC was the lowest (P<0.01) among all analyzed cells. It
increased dramatically in OPC (1.42-fold) and was maintained
at high levels in OL at 4 and 14DIV (Figure 4C). However, the
expression pattern of V2 was somewhat different from that
of V1/V0. The mRNA level of V2 in NPC was very low, peaked
in OPC (2.13 fold vs NPC, P<0.01), slightly decreased in OL at
4DIV, and markedly decreased in OL at 14DIV. The
expression of V2 in OL at 14DIV was significantly lower than that in
OPC (P<0.01) and OL at 4DIV (P<0.05), and showed no
significant difference with that in NPC (Figure 4D).
Regulation of versican expression in NPC by proinflammatory cytokines A previous report demonstrated
that TNF receptor I and IFN-γ receptor were expressed on
the NPC[31] making it possible to study
the effect of TNF-α and IFN-γ on NPC. To determine whether the expression
patterns of versican isoforms could be regulated by these 2
pro-inflammatory cytokines, NPC were treated for 48 h with
different doses of TNF-α, IFN-γ, or both, and collected for
relative quantitative real-time PCR. The melting curve
analysis (Figure 5A) and the agarose gel electrophoresis analysis
(Figure 5B) for V1, V2, and HPRT confirmed the specificity of
these amplified products.
The results revealed that the 2 pro-inflammatory
cyto-kines, used separately or in combination, did not affect the
expression of V1/V0, but obviously affected the V2 mRNA
expression in NPC in a dose-dependent manner (Figure
5C_5E). The 80 U/mL TNF-α could significantly increased
the level of V2 mRNA by 3.2-fold over the untreated
control (P<0.01), but low-dose TNF-α had no obvious effects
(Figure 5C). Similarly, treatment with 30, 70, and 100 U/mL
IFN-γ, could upregulate V2 mRNA levels by 1.8-
(P>0.05), 4.1- (P<0.01), and 2.9-fold
(P<0.01), respectively (Figure 5D). A combination of the 2 cytokines exhibited an additive effect
and the level of V2 mRNA was increased by 3.3-fold
(P<0.01) compared to 1.5-fold (P>0.05) with
TNF-α or 2.5-fold (P<0.01) with IFN-γ alone (Figure 5E).
Discussion
Versican expression in NPC and their
lineages Versican is a large chondroitin sulfate proteoglycan identified as one
of the major extracellular molecules in the brain and belongs
to the family of lecticans[32,33]. Previous reports showed that
versican was selectively expressed in embryonic tissues that
act as barriers to neural crest cell migration and axon
outgrowth[23,34]. In this study, we showed that versican was
expressed in NPC isolated from the embryonic rat spinal cord,
detected by both immunofluorescence double-staining and
RT-PCR. The expression of versican in NPC may be
related to the highly precise and coordinated migration of
neural crest cells during the early phase of embryonic
development.
Our ICC results also showed that versican was widely
located in cells differentiated from NPC, including neurons,
astrocytes, and oligodendrocytes. To strengthen these
results, we employed RT-PCR to verify the expression of
versican in neurons isolated from the E18 rat embryonic
cerebral cortex and cultured in vitro for 7 d. The results of
the RT-PCR completely coincided with the immunostaining
results showing that both V1/V0 and V2 mRNA were
expressed in neurons.
It has long been believed that versican is a glial product
based on its distribution in the normal and injured CNS. High
levels of versican were found in white matter tracts and it
was also readily detectable in the spinal cord gray matter,
but very little in the cerebral
cortex[8]. Our finding that versican was also expressed in neurons has added a new
dimension to previous findings and raised an intriguing
question as to what possible physiological role versican
may play in neurons during development or after injury.
Further, we also questioned whether the expression of
versican in neurons contributes to the formation of
peri-neuronal nets (PNN). PNN, a specialized form of
extracellular matrix around the cell body and dendrites of many classes
of neurons in the adult CNS, are composed of several
molecules including CSPG, hyaluronan, tenascin-R, and link
protein[35]. Some CSPG, such as aggrecan and versican,
members of the lectican family, are associated with PNN. These
molecules play a role in restricting the plastic capabilities of
adult neurons[36] and act as a protection for neurons against
oxidative stress. However, until now, the cellular sources of
PNN components remain unclear. In this work, we described
the expression of versican in neurons and glial cells by ICC
and/or RT-PCR, and the results suggested that both neurons
and glial cells might participate in synthesizing PNN
components. The neuronal expression of versican may
indicate that this molecule may play a role in maintaining synaptic
stability and preventing plasticity in the mature CNS. On the
other hand, the wide distribution of versican in neurons and
glial cells suggests that versican may be critical in the
interaction between matrices and these cells.
Versican expression in oligodendrocyte lineage
cells Previous in vitro studies revealed that versican was
mainly a product of oligodendrocyte lineage
cells[27]. In this study, based on high purity OPC generated from rat
embryonic NPC, an in vitro model which could mimic the
environment of oligodendrogliogenesis in
vivo was established. Therefore, we could conveniently obtain
oligodendrocyte lineage cells at different developmental stages
in vitro to elucidate the expression pattern of versican isoforms
during oligodendrogliogenesis. The ICC results showed that
versican was not only expressed in bipolar or tripolar
A2B5+ OPC, but also on the soma and processes of multipolar
O4+ pre-oligodendrocytes, more branched
O1+-immature oligodendrocytes, and even more differentiated
MBP+-myelin-forming oligodendrocytes. These results were mostly
in accordance with previous reports[27] except the
expression of versican on MBP+ oligoden-drocytes. However, other
investigators had detected the expression of versican in
MBP+ oligodendrocytes by in situ hybridization
in vivo[25]. We supposed that the difference in the methods used for OPC
induction might have brought up the discrepancy with the
reports.
Previous studies showed that different isoforms of
versican exhibited distinct and complementary expression
patterns in the developing CNS[22]. Versican V2 was present
at relatively low levels during the late embryonic stage and
further decreased by approximately 50% between 1 and 2
weeks' postnatal, then increased steadily to reach a
maximum at 100 d, 7-fold more that at 10 d
postnatal[37], whereas versican V1/V0 was mainly expressed in the late stages of
embryonic development[23], and its level doubled between
E14 and birth, after which V1/V0 decreased by 90% to reach
a low "mature" level that remained stable throughout
adulthood[37]. These observations and clear-cut changes
suggest that versican V1/V0 and V2 play different roles during
neurogenesis and homeostasis of the mature brain.
In this study, we examined the expression patterns of
different versican isoforms in the different developmental
stages of oligodendrocyte lineage cells. Our RT-PCR
results showed that embryonic spinal cord-derived NPC
expressed both versican V1/V0 and V2, but at relatively low
levels. Once they differentiated into oligodendrocyte
lineage cells, the expression of both isoforms increased
significantly. However, the expression patterns of V1/V0
and V2 were different during the course of OL differentiation.
From OPC to early (4DIV) and late OL (14DIV), the
expression of versican V1/V0 did not change significantly, whereas
that of versican V2 decreased markedly in OL at 14DIV
compared with OPC or OL at 4DIV. Notably, these expression
patterns of versican isoforms in oligodendrogliogenesis
in vitro are different from those in the developing brain as
mentioned above[37]. As the in
vivo study reflected, there was a complete change of versican expressional profiles in the
extracellular matrix and in all cell types, including neurons, OL
lineage cells, astrocytes, as well as non-neural cells, such as
meningeal cells[27]. This suggests that the expression
patterns of versican isoforms in different cells during CNS
development may be different. Further study of the expression
patterns of these isoforms in different cell types may be
beneficial for the understanding of the function of versican
isoforms in the developing CNS.
It has been recognized that different versican isoforms
may have distinct biological functions. For example, the
expression of versican V2 in the white matter and CNS
myelin suggests a role of this molecule in restricting structural
plasticity and regeneration of CNS fiber
tracts[25]. However, coculture experiments showed that versican V1/V0 had no
inhibitory effect on axon extension[38] and instead induced
neuronal differentiation and promoted neurite
outgrowth in vitro[26]. Thus, the complementary effects of V1/V0 and V2
on neurite outgrowth imply that the dynamically balanced
expression patterns of these isoforms may provide a
suitable extracellular environment for neurite development and
homeostasis of the mature brain.
In the development of CNS, the distribution of OPC is
consistent with their function in axonal guidance, perhaps
channeling axons and preventing them from straying into
inappropriate areas. Furthermore, OPC were present at
greater numbers during the last 2_3 d of gestation and the
first week of rat postnatal life[39], which is in accordance
with their function in preventing errors and axonal straying.
Versican V2, as well as other CSPG produced by OPC, such
as NG2, may also play a critical role during the development
of axonal pathways. From this point on, it was not
surprising that putatively inhibitory isoform versican V2 was
expressed at the highest level at the OPC stage. The results
presented here suggest that the surface of OPC might be
non-permissive for axon growth and repel growing axons.
However, in another observation made at our
laboratory, OPC/oligodendrocytes cocultured with dorsal root ganglion
(DRG) explants showed that OPC were less inhibitory to
neurite outgrowth of DRG neurons than mature oligodendrocytes
(Zheng-wen MA, personal communication). It implied that
the high-level expression of versican V2 in OPC might not be
parallel to their inhibitory function, and the inhibition effect of
OL lineage cells may be determined by a combination of a series
of inhibitory molecules, including Nogo, OMgp, and
Myelin-associated glycoprotein (MAG), not only by versican V2.
Effect of pro-inflammatory cytokines on versican
expression After injury to the adult CNS, one of the earliest
responses to injury is the infiltration of macrophages and the
activation of microglia, which starts a cytokine/growth
factor cascade[40]. Numerous growth factors and cytokines are
released that could potentially regulate the expression of
CSPG. Recently, it has been revealed that the presence of
TGF-β1 and EGF greatly increased the production of
several CSPG by astrocytes[41]. Moreover, it was reported that
TGF-β and IL-1β could bring about an increase in the amount
of versican in OPC and their differentiating cells,
respectively[27]. It was also found that versican V2 expression was
upregulated in response to brain injury and was suggested
to be induced, directly or indirectly, by cytokines and growth
factors released after injury[27]. In contrast, versican V1 and
V0 were not detected in the damaged tissue or in the
surrounding region after CNS
injury[38]. These results suggest that the expression of versican isoforms, especially V2, could
be regulated by cytokines and other molecules presented in
injured CNS environments.
It was reported that NPC had a tendency to migrate
towards damaged regions after CNS injury in
vivo[42,43], which was the first critical step in NPC engagement during
regeneration. In addition, evidence revealed that 2 other
cytokines, TNF-α and IFN-γ, increased after traumatic brain
injury[44] and spinal cord
injury[45_47]. Here we report that both
TNF-α and IFN-γ can upregulate the transcription of
versican V2 in NPC in a dose-dependent manner within a
certain dose range, yet had no effect on V1/V0 expression.
This result was in agreement with the phenomenon that
versican was upregulated in the injured CNS and provided
some explanations that TNF-α and IFN-γ are partly
responsible for the different expression changes of versican
isoforms after CNS injury. Combined with other
reports[27,41], it is reasonable to conclude that injury-induced changes in
cytokines might alter the expression pattern of CSPG and
several important inhibitory proteoglycans, such as versican
V2 and NG2[48] being overexpressed, therefore, causing
regenerative failure around the lesion sites. In this case, for
NPC transplantation to repair the CNS injury, some
measures need to be taken to modulate or reduce CSPG
production in the injured CNS prior to the transplantation.
Treatment with chondroitinase by degrading its
glycosaminoglycan components may abolish the inhibitory effect of versican
V2 and other inhibitory proteoglycans on neurite outgrowth
and promote axonal
regeneration[36,49,50].
In this study, we also found that after exposure to
IFN-γ, the floating neurospheres tended to attach to the bottom
of the flask. Some cells migrated from the spheres and
showed a significant tendency of differentiation. As we
revealed in this study, once NPC differentiate into
oligodendrocyte lineages, versican V2 expression increases
significantly, therefore, it is possible that the increase in
versican V2 expression was an indirect consequence of the
differentiation of NPC induced by pro-inflammatory
cytokines, rather than a direct effect of cytokines on NPC.
Certainly, the cases in vivo may be more complex and the
expression changes of versican isoforms upon these
cytokines on other cell types in the CNS remain to be
determined.
It has been revealed that a high dose of
pro-inflammatory cytokines could inhibit NPC proliferation and increase
apoptosis[31], and the change of cell vitality caused by
cytokines might have an effect on versican gene expression.
We also noted that the mRNA expression of versican V2
changed in a dose-dependent manner; within a certain dose
range, it raised steadily, but after it reached its peak, it quickly
declined. Therefore, to avoid the inhibitory effect of a high
dose of cytokines on the status or vitality of NPC, both
cytokines used in this study were restricted in medium
concentrations and within a narrow range.
In conclusion, the present study demonstrated that
versican was constitutively expressed in neurons, astrocytes,
and oligodendrocyte lineage cells differentiated from
NPC in vitro. By means of simple and efficient methods of OPC
induction and differentiation in vitro, we compared the
expression patterns of versican isoforms V1/V0 and V2 at
different developmental stages of oligodendrocyte
development from NPC. The results showed that the 2 versican
isoforms expressed throughout the oligodendrocyte
development stages, but inhibitory versican V2 expression peaked
at the OPC stage. We also found that pro-inflammatory
cytokines TNF-α and IFN-γ could upregulate the
transcription of inhibitory versican V2 in NPC in a dose-dependent
manner within a certain dose range, but had no effect on
V1/V0 expression, which might be partly related to the
formation of non-permissive surroundings for axonal
regeneration after CNS injuries in vivo.
Acknowledgements
The authors thank Prof Yi-ping ZHANG, Department of
Neurology, University of California, for the generous gift
of versican antibodies and valuable suggestions.
References
1 Ingber DE, Folkman J. Mechanochemical switching between
growth and differentiation during fibroblast growth
factor-stimulated angiogenesis in vitro: role of extracellular matrix. J Cell
Biol 1989; 109: 317_30.
2 Mongiat M, Fu J, Oldershaw R, Greenhalgh R, Gown AM, Iozzo
RV. Perlecan protein core interacts with extracellular matrix
protein 1 (ECM1), a glycoprotein involved in bone formation
and angiogenesis. J Biol Chem 2003; 278: 17 491_9.
3 Walker HA, Whitelock JM, Garl PJ, Nemenoff RA, Stenmark
KR, Weiser-Evans MC. Perlecan up-regulation of FRNK
suppresses smooth muscle cell proliferation via inhibition of FAK
signaling. Mol Biol Cell 2003; 14: 1941_52.
4 Oohira A, Matsui F, Matsuda M, Shoji R. Developmental change
in the glycosaminoglycan composition of the rat brain. J
Neurochem 1986; 47: 588_93.
5 Zimmermann DR, Ruoslahti E. Multiple domains of the large
fibroblast proteoglycan, versican. EMBO J 1989; 8: 2975_81.
6 Shinomura T, Nishida Y, Ito K, Kimata K. cDNA cloning of
PG-M, a large chondroitin sulfate proteoglycan expressed during
chondrogenesis in chick limb buds. Alternative spliced multiforms
of PG-M and their relationships to versican. J Biol Chem 1993;
268: 14 461_9.
7 Paulus W, Baur I, Dours-Zimmermann MT, Zimmermann DR.
Differential expression of versican isoforms in brain tumors. J
Neuropathol Exp Neurol 1996; 55: 528_33.
8 Bignami A, Perides G, Rahemtulla F. Versican, a
hyaluronate-binding proteoglycan of embryonal precartilaginous mesenchyma,
is mainly expressed postnatally in rat brain. J Neurosci Res
1993; 34: 97_106.
9 Ang LC, Zhang Y, Cao L, Yang BL, Young B, Kiani C,
et al. Versican enhances locomotion of astrocytoma cells and reduces
cell adhesion through its G1 domain. J Neuropathol Exp Neurol
1999; 58: 597_605.
10 Wight TN. Versican: a versatile extracellular matrix proteoglycan
in cell biology. Curr Opin Cell Biol 2002; 14: 617_23.
11 Zhang Y, Cao L, Yang BL, Yang BB. The G3 domain of versican
enhances cell proliferation via epidermal growth factor-like
motifs. J Biol Chem 1998; 273: 21 342_51.
12 Zhang Y, Cao L, Kiani CG, Yang BL, Yang BB. The G3 domain
of versican inhibits mesenchymal chondrogenesis via the
epidermal growth factor-like motifs. J Biol Chem 1998; 273: 33
054_63.
13 Zhang Y, Cao L, Kiani C, Yang BL, Hu W, Yang BB. Promotion
of chondrocyte proliferation by versican mediated by G1 domain
and EGF-like motifs. J Cell Biochem 1999; 73: 445_57.
14 Ito K, Shinomura T, Zako M, Ujita M, Kimata K. Multiple
forms of mouse PG-M, a large chondroitin sulfate proteoglycan
generated by alternative splicing. J Biol Chem 1995; 270:
958_65.
15 Lemire JM, Braun KR, Maurel P, Kaplan ED, Schwartz SM, Wight
TN. Versican/PG-M isoforms in vascular smooth muscle cells.
Arterioscler Thromb Vasc Biol 1999; 19: 1630_9.
16 Aspberg A, Binkert C, Ruoslahti E. The versican C-type lectin
domain recognizes the adhesion protein tenascin-R. Proc Natl
Acad Sci USA 1995; 92: 10 590_4.
17 Isogai Z, Aspberg A, Keene DR, Ono RN, Reinhardt DP, Sakai
LY. Versican interacts with fibrillin-1 and links extracellular
microfibrils to other connective tissue networks. J Biol Chem
2002; 277: 4565_72.
18 Kawashima H, Hirose M, Hirose J, Nagakubo D, Plaas AH,
Miyasaka M. Binding of a large chondroitin sulfate/dermatan
sulfate proteoglycan, versican, to L-selectin, P-selectin, and
CD44. J Biol Chem 2000; 275: 35 448_56.
19 Matsumoto K, Shionyu M, Go M, Shimizu K, Shinomura T,
Kimata K, et al. Distinct interaction of versican/PG-M with
hyaluronan and link protein. J Biol Chem 2003; 278: 41
205_12.
20 LeBaron RG, Zimmermann DR, Ruoslahti E. Hyaluronate
binding properties of versican. J Biol Chem 1992; 267: 10 003_10.
21 Olin AI, Morgelin M, Sasaki T, Timpl R, Heinegard D, Aspberg
A. The proteoglycans aggrecan and versican form networks with
fibulin-2 through their lectin domain binding. J Biol Chem 2001; 276:
1253_61.
22 Bandtlow CE, Zimmermann DR. Proteoglycans in the
developing brain: new conceptual insights for old proteins. Physiol Rev
2000; 80: 1267_90.
23 Landolt RM, Vaughan L, Winterhalter KH, Zimmermann DR.
Versican is selectively expressed in embryonic tissues that act as
barriers to neural crest cell migration and axon outgrowth.
Development 1995; 121: 2303_12.
24 Schmalfeldt M, Dours-Zimmermann MT, Winterhalter KH,
Zimmermann DR. Versican V2 is a major extracellular matrix
component of the mature bovine brain. J Biol Chem 1998; 273:
15 758_64.
25 Schmalfeldt M, Bandtlow CE, Dours-Zimmermann MT,
Winterhalter KH, Zimmermann DR. Brain derived versican V2
is a potent inhibitor of axonal growth. J Cell Sci 2000; 113:
807_16.
26 Wu Y, Sheng W, Chen L, Dong H, Lee V, Lu F,
et al. Versican V1 isoform induces neuronal differentiation and promotes neurite
outgrowth. Mol Biol Cell 2004; 15: 2093_104.
27 Asher RA, Morgenstern DA, Shearer MC, Adcock KH, Pesheva
P, Fawcett JW. Versican is upregulated in CNS injury and is a
product of oligodendrocyte lineage cells. J Neurosci 2002; 22:
2225_36.
28 Hu JG, Fu SL, Zhang KH, Li Y, Yin L, Lu PH,
et al. Differential gene expression in neural stem cells and oligodendrocyte
precursor cells: a cDNA microarray analysis. J Neurosci Res 2004; 78:
637_46.
29 Fu SL, Hu JG, Li Y, Yin L, Jin JQ, Xu XM,
et al. Induction of rat neural stem cells into oligodendrocyte precursor cells. Acta
Physiol Sin 2005; 57: 132_8.
30 Zhang SC, Lundberg C, Lipsitz D, O'Connor LT, Duncan ID.
Generation of ligodendroglial progenitors from neural stem cells.
J Neurocytol 1998; 27: 475_89.
3 1 Ben-Hur T, Ben-Menachem O, Furer V, Einstein O,
Mizrachi-Kol R, Grigoriadis N. Effects of proinflammatory cytokines on
the growth, fate, and motility of multipotential neural precursor
cells. Mol Cell Neurosci 2003; 24: 623_31.
32 Aspberg A, Miura R, Bourdoulous S, Shimonaka M, Heinegard D,
Schachner M, et al. The C-type lectin domains of lecticans, a
family of aggregating chondroitin sulfate proteoglycans, bind
tenascin-R by protein-protein interactions independent of
carbohydrate moiety. Proc Natl Acad Sci USA 1997; 94: 10
116_21.
3 3 Yamaguchi Y. Lecticans: organizers of the brain extracellular matrix.
Cell Mol Life Sci 2000; 57: 276_89.
34 Dutt S, Kleber M, Matasci M, Sommer L, Zimmermann DR.
Versican V0 and V1 guide migratory neural crest cells. J Biol
Chem 2006; 281: 12: 123_31.
35 Celio MR, Spreafico R, De BS, Vitellaro-Zuccarello L.
Perineu-ronal nets: past and present. Trends Neurosci 1998; 21: 510_5.
36 Pizzorusso T, Medini P, Berardi N, Chierzi S, Fawcett JW, Maffei
L. Reactivation of ocular dominance plasticity in the adult
visual cortex. Science 2002; 298: 1248_51.
37 Milev P, Maurel P, Chiba A, Mevissen M, Popp S, Yamaguchi Y,
et al. Differential regulation of expression of
hyaluronan-binding proteoglycans in developing brain: aggrecan, versican,
neurocan, and brevican. Biochem Biophys Res Commun 1998;
247: 207_12.
38 Fidler PS, Schuette K, Asher RA, Dobbertin A, Thornton SR,
Calle-Patino Y, et al. Comparing astrocytic cell lines that are
inhibitory or permissive for axon growth: the major
axon-inhibitory proteoglycan is NG2. J Neurosci 1999; 19: 8778_88.
39 Levine JM, Stincone F, Lee YS. Development and differentiation of
glial precursor cells in the rat cerebellum. Glia 1993; 7: 307_21.
40 Giulian D, Chen J, Ingeman JE, George JK, Noponen M. The
role of mononuclear phagocytes in wound healing after
traumatic injury to adult mammalian brain. J Neurosci 1989; 9:
4416_29.
41 Smith GM, Strunz C. Growth factor and cytokine regulation of
chondroitin sulfate proteoglycans by astrocytes. Glia 2005; 52:
209_18.
42 Boockvar JA, Schouten J, Royo N, Millard M, Spangler Z,
Castelbuono D, et al. Experimental traumatic brain injury
modulates the survival, migration, and terminal phenotype of
transplanted epidermal growth factor receptor-activated neural stem
cells. Neurosurgery 2005; 56: 163_71.
43 Imitola J, Raddassi K, Park KI, Mueller FJ, Nieto M, Teng YD,
et al. Directed migration of neural stem cells to sites of CNS injury
by the stromal cell-derived factor 1alpha/CXC chemokine
receptor 4 pathway. Proc Natl Acad Sci USA 2004; 101: 18
117_22.
44 Fan L, Young PR, Barone FC, Feuerstein GZ, Smith DH,
McIntosh TK. Experimental brain injury induces differential
expression of tumor necrosis factor-alpha mRNA in the CNS. Brain
Res Mol Brain Res 1996; 36: 287_91.
45 Hayashi M, Ueyama T, Nemoto K, Tamaki T, Senba E.
Sequential mRNA expression for immediate early genes, cytokines, and
neurotrophins in spinal cord injury. J Neurotrauma 2000; 17:
203_18.
46 Nakamura M, Houghtling RA, MacArthur L, Bayer BM, Bregman
BS. Differences in cytokine gene expression profile between
acute and secondary injury in adult rat spinal cord. Exp Neurol
2003; 184: 313_25.
47 Yan P, Li Q, Kim GM, Xu J, Hsu CY, Xu XM. Cellular
localization of tumor necrosis factor-alpha following acute spinal cord
injury in adult rats. J Neurotrauma 2001; 18: 563_8.
48 Jones LL, Yamaguchi Y, Stallcup WB, Tuszynski MH. NG2 is a
major chondroitin sulfate proteoglycan produced after spinal cord
injury and is expressed by macrophages and oligodendrocyte
progenitors. J Neurosci 2002; 22: 2792_803.
49 Bradbury EJ, Moon LD, Popat RJ, King VR, Bennett GS, Patel
PN, et al. Chondroitinase ABC promotes functional recovery
after spinal cord injury. Nature 2002; 416: 636_40.
50 Chau CH, Shum DK, Li H, Pei J, Lui YY, Wirthlin L,
et al. Chondroitinase ABC enhances axonal regrowth through Schwann
cell-seeded guidance channels after spinal cord injury. FASEB J
2004; 18: 194_6.
|
