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Short Communications |
From the Division of Neurology,*
Department of Internal
Medicine, Saga Medical School, Saga; and the Department of
Bacteriology,
Nagasaki University School of
Medicine, Nagasaki, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, five independent groups have established lines of mice devoid of the PrPC protein (PrP-/-), designated Zur, Npu, Ngsk, Rcm0, and Rikn, using different gene-targeting strategies.6-13 The entire open reading frame of the PrP gene (Prnp) was replaced by selectable markers in Ngsk, Rcm0, and Rikn PrP-/- mice, whereas a part of the open reading frame remained intact in Zur and Npu PrP-/- mice. All of these mice exhibited normal early development and complete protection against scrapie infection, indicating that PrPC, a dispensable protein in embryonic development, is essential for inducing prion diseases.6-12 Zur PrP-/- mice showed impairment in the GABAA receptor-mediated fast inhibition and long-term potentiation in hippocampal CA1 pyramidal neurons, suggesting that PrPC is necessary for normal synaptic function and plasticity in the hippocampus.14,15 However, some investigators were unable to confirm these observations.16 Both Zur and Npu PrP-/- mice exhibited altered circadian activity rhythms and sleep patterns.17 Ngsk, Rcm0, and Rikn PrP-/- mice exhibited late-onset cerebellar ataxia due to an extensive loss of Purkinje cells in the cerebellum.10-12 Furthermore, Ngsk PrP-/- mice showed a significant amount of demyelination in the spinal cord and peripheral nerves, although the pathophysiological basis for these abnormalities remains unclear.10-12 The introduction of a wild-type PrP transgene rescued them from Purkinje cell degeneration and demyelination, indicating that PrPC is directly involved not only in the long-term survival of Purkinje neurons but also in the myelinating capacity of oligodendrocytes and Schwann cells.10,11
The cDNA array technology is a novel approach that allows monitoring of the expression pattern of a large number of genes systematically in a single hybridization, using cDNA probes prepared from different RNA sources on a matrix where a wide variety of cDNA fragments or oligonucleotides are arrayed and immobilized.18,19 Previously, using Western blot analysis, we showed that the constitutive and heat-inducible expression of heat shock proteins, HSP105, HSP72, HSC70, HSP60, and HSP25 was similar between two distinct fibroblast cell lines isolated from Ngsk PrP-/- mice and the control PrP+/+ mice, suggesting that the PrPC protein may not act as a cellular regulator during a heat shock response.20 In this study, the gene expression profile was studied in these cell lines by analyzing a cDNA expression array containing 597 clones of various functional classes to elucidate the physiological function of the PrPC protein.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The method for producing mice homozygous for a disrupted PrP gene (Ngsk PrP-/- mice) was previously described.9-11 Three distinct fibroblast cell lines were established from abdominal skin explant cultures of the homozygous PrP-/- mice with a mixed 129/Sv x C57BL/6J background (SFK), the heterozygous PrP+/- mice (SFHT), and the control C57BL/6J PrP+/+ mice (SFH) at ages 35 to 50 weeks, as described previously.20 Both PrP-/- and PrP+/- mice were produced by intercross between F1 PrP+/- breeding pairs and their genotypes were determined by Southern blot analysis,9-11 whereas PrP+/+ mice were obtained from Kyudo (Kumamoto, Japan). The cells were plated in 25-cm2 culture flasks at a density of 106 cells/flask and incubated at 37°C in a 5% CO2/95% air incubator in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 µg/ml of streptomycin (feeding medium). The passage of the cultures was performed biweekly. They were maintained for 4 months (SFK, SFH, and SFHT) or 7 months (SFK) before starting the experiments. After the cultures became subconfluent, the culture medium was replaced by a feeding medium containing 1% FBS instead of 10% FBS; this is the low serum concentration (LS) medium. After a 72-hour incubation in LS medium with or without inclusion of 50 ng/ml of recombinant human basic fibroblast growth factor (bFGF; Sigma, St. Louis, MO) for the final 24 hours, they were processed for RNA preparation.
Extraction of RNA and Synthesis of 32P-Labeled cDNA Probes
Total RNA was extracted from bFGF-treated SFK, SFH, and SFHT cells
(SFK-B, SFH-B, and SFHT-B) and untreated SFK, SFH, and SFHT cells
(SFK-C, SFH-C, and SFHT-C), or from the whole cerebral cortices
isolated from the PrP-/-
mice (CBRK) and from the PrP+/+ mice (CBRH), from
which SFK or SFH cells have originated. Poly A+
RNA was purified from total RNA pretreated with DNase I, using the
oligotex-dT30 latex bead system (Takara, Tokyo, Japan). One microgram
of poly A+ RNA was reverse-transcribed by
incubating at 50°C for 20 minutes in 11 µl of a reaction mixture
containing 50 mmol/L Tris-HCl, pH 8.3, 75 mmol/L KCl, 3 mmol/L
MgCl2, 500 µmol/L dCTP, 500 µmol/L dGTP, 500
µmol/L dTTP, 5 µmol/L dATP, 35 µCi of
[
-32P]dATP, 5 mmol/L DTT, 50 units of
Moloney murine leukemia virus (MMLV) reverse transcriptase, and the
coding sequence primers at a final concentration of 1x using a
commercial kit (Clontech, Palo Alto, CA) followed by purification
through column chromatography.
Southern Blot Hybridization of a cDNA Expression Array
A set of filters containing 597 mouse cDNA clones of various functional classes immobilized as a spot of duplicate dots on a positively charged nylon membrane (Atlas Mouse cDNA Expression Array I, Clontech) were hybridized at 68°C overnight in the hybridization solution containing the 32P-labeled cDNA probes at a concentration of 1 x 106 cpm/ml, according to the methods described previously.21 The membranes were exposed to Kodak BioMax MS X-ray films with the intensifying screen at -80°C for 24 hours. For rehybridization, the probes were stripped from the membranes by washing in boiled 0.5% sodium dodecyl sulfate solution for 15 minutes. Densitometric analysis was performed on an imaging system using NIH Image version 1.61 software. The signal intensities were standardized against those of a housekeeping gene, ß-actin.
RT-PCR Analysis and Northern Blot Analysis
For reverse transcription-polymerase chain reaction (RT-PCR)
analysis, 5 µg of total RNA pretreated with DNase I was processed for
cDNA synthesis using oligo(dT)12–18 primers and
SuperScript II reverse transcriptase (Gibco BRL, Gaithersburg, MD).
Fifty nanograms of cDNA were amplified by PCR using the specific sense
and antisense primers listed in Table 1
. For Northern blot analysis, 2 µg
(SFK, SFH, and SFHT) or 6 µg (CBRK and CBRH) of total RNA was
separated on a 1.5% agarose-6% formaldehyde gel, transferred onto
nylon membranes, and immobilized by UV fixation as described
previously.21,22
The membranes were hybridized at 53°C
overnight in the hybridization buffer containing a digoxigenin
(DIG)-labeled DNA probe synthesized using a PCR DIG probe synthesis kit
(Boehringer Mannheim, Mannheim, Germany) and the specific sense and
antisense primers listed in Table 1
. The membranes were further
processed for rehybridization with the DIG-labeled DNA probe specific
for the ß-actin gene to normalize the reaction. The specific reaction
was visualized using the DIG chemiluminescence detection kit
(Boehringer Mannheim).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With RT-PCR analysis, the expression of PrP mRNA was undetectable
in bFGF-treated and untreated SFK cells (SFK-B and SFK-C), whereas it
was identified in SFH cells derived from the
PrP+/+ mice under both culture conditions, SFH-B
and SFH-C (Figure 1
, lanes 2, 4, 6, and
8). In contrast, the expression of ß-actin mRNA was observed in both
SFK and SFH cells (Figure 1
, lanes 10, 12, 14, and 16). No products
were amplified in total RNA samples processed for PCR when the reverse
transcription step was omitted, confirming that a contamination of
genomic DNA was excluded (Figure 1
, lanes 1, 3, 5, 7, 9, 11, 13, and
15).
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The cDNA expression array was analyzed by Southern blot
hybridization with the 32P-labeled cDNA probes
prepared from poly A+ RNA which was isolated from
SFH-C, SFK-C, SFH-B or SFK-B cells, all of which were maintained for 4
months in vitro. Among the 597 cDNA clones, 42 genes
exhibited a differential expression pattern between SFK-C and SFH-C
cells (Figure 2, a and b
, and Table 2
). The expression of 15 genes including
those essential for cell proliferation and adhesion, such as
c-myc proto-oncogene, cyclin D1, the receptor tyrosine
kinase substrate Eps8, CD44, and vascular cell adhesion molecule-1, was
reduced in SFK-C cells compared to the levels in SFH-C cells (Table 2)
.
In contrast, the expression of 27 genes including those involved in the
insulin-like growth factor-I (IGF-I) signaling pathway, such as
phosphatidylinositol 3-kinase (PI3K) p85, IGF-binding proteins (IGFBP)
-3, -5, and -6, and IGF-I, was elevated in SFK-C cells compared to the
levels in SFH-C cells (Table 2)
. Exposure to bFGF elevated the
expression levels of 25 genes in SFH cells and 17 genes in SFK cells by
up-regulating eight genes shared between both, such as 14-3-3
and
the UV excision repair protein homologue MHR23B (Figure 2, a
-d, and
Table 2
). Treatment with bFGF reduced the levels of expression of 15
genes in SFH cells and 22 genes in SFK cells by down-regulating 12
genes shared between both, such as cdk4/cdk6 inhibitor p18 and protein
kinase C-
(Figure 2, a
-d, and Table 2
).
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Since it is impractical to quantify all 42 genes expressed
differentially between SFK and SFH cells under untreated conditions
using Northern blot analysis, six clones exhibiting a great difference
between both were selected in view of their potential involvement in
cell proliferation, differentiation, and survival. They included three
genes down-regulated in SFK-C cells versus SFH-C cells: Eps8
(Figure 2
, spot 1), cyclin D1 (Figure 2
, spot 2), and CD44 (Figure 2
,
spot 3) and three genes up-regulated in SFK-C cells versus
SFH-C cells: PI3K p85 (Figure 2
, spot 4), IGF-I (Figure 2
, spot 5), and
the serine protease inhibitor (Spi)-2.2, a mouse equivalent to the
human 1-antichymotrypsin (ACT)23
(Figure 2
, spot 6). In
Northern blotting, the levels of expression of Eps8 (4.7 kb), cyclin D1
(4.5 and 3.8 kb), and CD44 (4.7 kb) mRNAs were reduced in SFK-C cells
to 33%, 28%, or 22% of those in SFH-C cells, respectively, when
standardized against corresponding ß-actin signals detected on the
identical blots (Figure 3A, a
-c, lanes 1
and 2). After exposure to bFGF, the levels of Eps8, cyclin D1, and CD44
mRNAs were elevated in SFH-B cells by 3.2-fold, 1.4-fold, or 1.9-fold,
respectively, compared to those in SFH-C cells, and increased in SFK-B
cells by 2.4-fold, 1.3-fold, or 2.1-fold, respectively, compared to
those in SFK-C cells (Figure 3A, a
-c, lanes 1–4). However, the levels
of Eps8, cyclin D1, and CD44 mRNAs were much lower in SFK-B cells than
those in SFH-C cells (Figure 3A, a
-c, lanes 1–4). In contrast, the
levels of PI3K p85 (7.0 and 4.0 kb), IGF-I (7.0, 1.6, and 0.8 kb) and
Spi-2.2 (2.1 kb) mRNAs were elevated in SFK-C cells by 4.6-fold,
6.0-fold, or 25.7-fold, respectively, compared to those in SFH-C cells
(Figure 3A, d
-f, lanes 1 and 2). After bFGF exposure, the expression of
PI3K p85, IGF-I and Spi-2.2 mRNAs was reduced in SFK-B cells to 59%,
85%, or 6% of those in SFK-C cells, and decreased in SFH-B cells to
71%, 17%, or 16% of those in SFH-C cells, respectively, with the
greatest reduction of Spi-2.2 expression in SFK cells after bFGF
treatment (Figure 3A, d
-f, lanes 1–4).
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To examine the possible effects of strain differences (129/Sv
x C57BL/6J background in SFK versus C57BL/6J background in
SFH) on the gene expression levels, a distinct cell line designated
SFHT was established from the skin explant cultures of the heterozygous
PrP+/- mice that were produced by intercross
between F1 PrP+/- breeding
pairs.9-11
The expression of Eps8, cyclin D1, CD44, PI3K
p85, IGF-I, and Spi-2.2 mRNAs was studied between bFGF-treated or
untreated SFHT cells (SFHT-B and SFHT-C) maintained for 4 months
in vitro and SFK cells (SFK-B and SFK-C) maintained for 7
months in vitro (Figure 3B, a
-f, lanes 1–4). In Northern
blotting, the patterns of expression of these mRNAs were almost
identical between SFK cells maintained for 4 months and those
maintained for 7 months in vitro (Figure 3A, a
-f, and 3B,
a-f, lanes 2 and 4), indicating that the culture periods might not
constitute a major factor contributing to the aberrant gene expression
identified in SFK cells. Furthermore, there was a high degree of
similarity between the patterns of expression of these mRNAs in SFHT-C
and SFK-C cells and those in SFH-C and SFK-C cells examined in the
initial experiments, except that SFHT-C cells expressed a lower level
of CD44 mRNA and a higher level of PI3K p85 mRNA than SFH-C cells
(Figure 3A, a
-f, and 3B, a-f, lanes 1 and 2). The levels of expression
of Eps8, cyclin D1, and CD44 mRNAs in SFK-C cells maintained for 7
months were reduced to 6%, 9%, or 34% of those in SFHT-C cells,
respectively (Figure 3B, a
-c, lanes 1 and 2). In contrast, the levels
of IGF-I and Spi-2.2 mRNAs were elevated in SFK-C cells by 4.9-fold or
4.2-fold, respectively, compared to those in SFHT-C cells (Figure 3B, e and f
, lanes 1 and 2). PrP mRNA was undetectable in SFK cells but
detectable in SFHT cells (Figure 3B
e, lanes 1–4). These results
suggest that the aberrant gene expression identified in SFK cells is
unlikely to be due to the effects of strain differences.
Differential Gene Expression in Brain Tissues of the PrP-/- Mice and PrP+/+ Mice
To evaluate the possibility that the differential gene expression
between SFK and SFH cells might represent a fibroblast cell
line-specific observation, the expression of Eps8, cyclin D1, CD44,
PI3K p85, IGF-I, and Spi-2.2 mRNAs was studied in the brain tissues of
the cerebrum isolated from the
PrP-/- mice (CBRK) and
the PrP+/+ mice (CBRH) from which SFK or SFH
cells originated. In Northern blotting, the levels of expression of
Eps8 and CD44 mRNAs in CBRK were reduced to 20% or 18% of those in
CBRH, when standardized against corresponding ß-actin signals
detected on the identical blots (Figure 3C, a and c
, lanes 1 and 2).
Both PI3K p85 and Spi-2.2 mRNA levels in CBRK were decreased slightly
to 83% or 88% of those in CBRH, whereas cyclin D1 mRNA expression was
not significantly different between both (Figure 3C, b, d, and f
, lanes
1 and 2). IGF-I mRNA was identified in neither of them, whereas PrP
mRNA was detectable in CBRH but undetectable in CBRK (Figure 3C
e, lanes
1 and 2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Ras proteins, activated after cell exposure to growth factors,
integrate a number of downstream effectors, leading to an activation of
the Raf/MAP kinase pathway, the Ral-GDS pathway, and the PI3K
pathway.24
Eps8 is originally identified as a widely
expressed substrate for the receptor tyrosine kinases
(RTKs).25
Its overexpression in cultured fibroblasts
enhances mitogenic responsiveness to endothelial growth
factor.25
Eps8 mRNA and protein expression is
induced markedly in cultured fibroblasts by exposure to serum or
phorbol esters, whereas it is suppressed in C2C12 myoblast cells after
terminal differentiation.26
Eps8 has a Src homology region
3 (SH3) domain that functions as an adapter, assembling intracellular
signal-transducing molecules with RTKs.25
Eps8 plays a
pivotal role in signal transduction from both Ras and PI3K to Rac, a
member of the subfamily of Rho-GTPases.27
The Rac proteins
that constitute the NADPH oxidase complex and act as a regulator of
actin cytoskeletal reorganization have a role in cell cycle progression
by activating the cyclin D1 promoter after the generation of
intracellular reactive oxygen species through NF-
B
activation.28
Cyclin D1 expression is up-regulated in MCF7
human breast cancer cells by exposure to IGF-I, followed by
hyperphosphorylation of a retinoblastoma protein Rb, whereas it is
suppressed by PI3K-specific inhibitors.29
CD44, a cell
surface glycoprotein expressed in both hematopoietic and
nonhematopoietic cell types, is involved in cell adhesion by
interacting with the extracellular matrix glycosaminoglycans
fibronectin, laminin, and collagen. The expression of CD44 protein or
its alternatively spliced variants is elevated in certain tumor cells
with metastatic potential and in Ras-transformed fibroblasts in
culture.30
All of these observations could support the
hypothesis that the down-regulation of Eps8, cyclin D1, and CD44 in SFK
cells might reflect a functional impairment in the Ras and Rac
signaling pathways in these cells.
The PI3K protein is composed of a 110-kd catalytic subunit (p110) and an 85-kd regulatory subunit (p85), the latter containing one SH3 domain and two SH2 domains by which it links to multiple signaling components. The PI3K protein, located immediately downstream from Ras, plays a role in insulin-stimulated glucose transport, exocytosis, neurite outgrowth, prevention of apoptosis, and cell cycle progression.24 The downstream targets of PI3K are the Rac proteins, the ribosomal protein kinase p70S6K, and the serine/threonine protein kinase PKB/Akt. IGF-I activates PI3K, followed by activation of PKB/Akt, which promotes IGF-I-dependent survival of cerebellar neurons in culture.31 Spi-2.2, a mouse homologue of the human ACT, belongs to a multigene family composed of closely related Spi-encoding genes derived by ancestral gene duplication. The expression of both Spi-2.1 and Spi-2.3 genes are up-regulated by growth hormone (GH) or by IGF-I, whereas Spi-2.2 gene expression is not GH-dependent but is induced after acute inflammatory reactions through activation of a panel of signal transducers and activators of transcription (STAT) proteins.23,32 IGF-I stimulates the release of ACT in MCF7 cells.33 All of these observations put forth a possible scenario that the up-regulated expression of PI3K p85, IGF-I, and Spi-2.2 mRNAs in SFK cells might represent a compensatory response to the reduced levels of cyclin D1 to maintain the cellular biological functions mediated by the IGF-I signaling pathway.34 The regulatory mechanisms underlying IGF-I gene expression are highly complex, and include the use of alternative promoters acting on multiple initiation sites, differential RNA splicing, polyadenylation, and translation.35 The biological activities of IGF-I are regulated by its half-life and affinity for specific receptors, which are affected by IGFBPs.35 IGFBPs are cleaved by a family of serine proteases into non-IGF-I-binding fragments.36 It is worth noting that the expression of a panel of IGFBP mRNAs is found to be elevated in SFK cells.
The mechanisms by which an absence of the PrPC protein in SFK cells deregulates the expression of a battery of genes located in the Ras and Rac signaling pathways remain unknown. The PrPC protein is enriched in a specialized compartment consisting of detergent-insoluble, cholesterol-rich membranous microdomains called caveolae-like domains (CLDs) or rafts, where a battery of signaling molecules including platelet-derived growth factor and endothelial growth factor receptors, Ras, PI3K, and nitric oxide synthase are clustered.37,38 PrPC interacts with PrPSc in this compartment during the formation of nascent PrPSc protein.37,38 PrP-deficient cells in culture showed an inability to deal with oxidative stress due to reduced activity of Cu/Zn superoxide dismutase, suggesting that the function of PrPC is important for cellular resistance to oxidative stress.4,39 A recent study using hippocampal neuronal progenitor cell lines isolated from Rikn PrP-/- mice has revealed that serum removal from these cultures facilitates apoptosis.13 Another study has shown that neuronal nitric oxide synthase is lost from the rafts in cerebellar tissues of adult Zur PrP-/- mice.40 All of these observations suggest that PrPC might play a role in the organization of signaling complexes in CLDs (rafts), and the deficiency of its function might disturb the CLD- (raft-) associated signal transduction that is pivotal for protection against oxidative stress and apoptosis, or for synaptic transmission, although further studies are required to evaluate this hypothesis.
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Acknowledgements |
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Footnotes |
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Supported in part by a grant from the Ichiro Kanehara Foundation.
Accepted for publication April 3, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antichymotrypsin-like proteins in a murine chondrocytic cell line. Gene 1991, 106:213-220[Medline]
1-antichymotrypsin and soluble IGF-II/mannose 6-phosphate receptor from MCF7 breast cancer cells. Endocrinology 1995, 136:3759-3766[Abstract]
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