|
http://www.abbs.info e-mail:[email protected] ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(4): 418-422 CN 31-1300/Q |
(
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences,
the Chinese Academy of Sciences, Shanghai 200031,
China )
Recently
it was found that in rat hippocampus, AVP4-8
could upregulate the CTP: phosphocholine cytidylyltransferase (CCT) which
catalyzed the rate-limiting step in the biosynthesis of phosphatidylcholine
(PC)[13]. PC is the major phospholipid in eukayotic cells and
function not only as an important structural component but also a major source
of second messengers for signal transduction[14, 15]. The product of
CCT reaction, CDP-choline, had been shown to have a therapeutic
effect on neurodegenerative disorders such as Alzheimer's disease (AD)[16].
This result could help us to elucidate the molecular basis of AVP4-8's
brain function and associate AVP4-8
with a promising drug for AD. As two mechanisms might contribute to mRNA
upregulation: acceleration of transcriptional
rate or stabilization of mRNA. In this report, we selected hippocampal neuron an in vitro research
system to investigate the main mechanism by which AVP4-8
regulates CCT mRNA in rat hippocampus and whether CCT activity was also
changed.
1.1
Materials
Sprague-Dawley
rats ( grade Ⅱ, certification No.003 ) were from
Shanghai Experimental Animal Center,
the Chinese Academy of Science. DMEM, fetal calf serum, B27 from Gibco
BRL, USA;[a-32P]-dCTP, [14C]-phosphocholine, [14C]-CDP-choline
from Amersham Pharmacia Biotech,
England; Actinomycin D,
phosphatidylcholine,
phosphocholine, oleic acid
from Sigma, USA; Prime-a-Gene
labeling system from Promega, USA;
K6 silica gel 60 plate from Whatmann,
England; Enhancer sprayer from NEN, USA; AVP4-8, ZDC(C)PR were synthesized and purified
by HPLC in our lab. All other reagents were of analytic or biochemical grade.
1.2
Cell incubation and drug treatment
Pregnant
SD rat was anaesthetizad with ether on day 17 gestation. Fetuses were removed
aseptically and the fetal hippocampi were dissected out in cold HBSS. The
neurons were isolated as described by reference [17]. After
incubated in DMEM containing 2% B27 supplement for at least 7 day, the cells were treated with drugs and
used for experiments.
1.3
RNA isolation and RT-PCR
Total
RNA was extracted by the acid guanidi-nium thiocyanate/phenol/chloroform method[18]
, and 2 mg
RNA was used to prepare cDNA using M-MuLV reverse transcriptase. The cDNAs were
then amplified using PCR. The PCR products were seperated by electrophoresis on
a 1% agarose gel, transferred to a
nylon membrane and fixed by baked at 80 ℃
for 2 h. The blots were hybridized with radiolabeled probes by the method of
Sambrook[19]. The membrane was exposed to X-ray film with
intensifying screen at -70 ℃.
PCR amplification was carried out with 20 cycles for CCT and 16 cycles for
GAPDH. The primers chosen for amplification of CCT were 5′-primer: 5′-ACG
TTT ATA AGC ATA TCA AG-3′, complementary to nucleotides 549-569;
and 3′-primer: 5′-TAA
GGC CTG TAG CAT CCG GA- 3′, corresponding to nucleotides 955-935.
The primers for GAPDH were 5′-primer: 5′-CTG
GAG AAA CCT GCC AAG TAT G-3′, and 3′-primer: 5′-CAC
CCT GTT GCT GTA GCC ATA-3′.
1.4
CCT assay
CCT
activity was determined by measuring the rate of incorporation of [14C]-phosphocholine
into CDP-choline. Each reaction mixture contained 4 mmol/L CTP,
10 mmol/L MgCl2,
150 mmol/L bis-tris-HCl ( pH 6.5 ), 1 mmol/L phosphocholine , 64 mmol/L
lipid activator ( PtdCho∶oleic
acid = 1∶1
), 7.4 kBq [14C]-phosphocholine
(specific activity, 2.0 gBq/mmol)
in a total assay volume of 50 ml.
The reaction was initiated by the addition of 50 mg
extracted neuron protein,
proceeded for 30 min at 37 ℃
and terminated by addition of 5 ml
of edetic acid 0.5 mol/L. Next, 20
ml
of each sample was spoted on preabsorbent silica gel G thin layer plates, which were developed in 2 % ammonium
hydroxide / 95 % ethanol ( 1/1 ). The plates were sprayed with an
autoradiographic enhancer sprayer and exposed to film for 7 day and then the
films were analysed densitometrically. CDP-[14C]-choline was
identified by co-migration with a standard.
2.1
Effects of AVP4-8
on the CCT mRNA of hippocampal neurons
After
incubated in DMEM containing 2 % B27 supplement for at least 7 day, rat hippocampal neurons were treated
with 10-7 mol/L AVP4-8 for 0, 3, 6, 9 and 12 h, then total RNA was isolated and used for RT-PCR and Southern
blot. As GAPDH mRNA in hippocampal neuons did not change with AVP4-8
incubation, RT-PCR for GAPDH was
carried out to test the RNA integrity and the efficiency of the
reverse-transcriptase reaction of each sample. Southern analysis revealed that
CCT mRNA increased corresponding to the AVP4-8
incubation(Fig.1). The statistic results of CCT/GAPDH ratio for 0, 3, 6, 9, 12
h are 1.00±0.04,
1.19±0.11b, 2.24±0.15c,
1.60±0.13c, 1.28±0.11b
respectively ( n = 4, the ratio of 0 h was chosen as control, cP < 0.01, bP < 0.05 vs
control). There was a 124 % increase in the relative amount of CCT mRNA in 6 h.
This upregulation could be greatly inhibited in the presence of 50-fold
ZDC(C)PR, the antagonist of AVP4-8
(Fig.2) , the CCT/GAPDH ratio of
AVP4-8
plus ZDC(C)PR (6 h) was reduced to 1.28±0.18f, [n = 4, fP < 0.01 vs AVP4-8
(6 h)].
The neurons were treated with AVP4-8
for (A) 0 h; (B) 3 h; (C) 6 h; (D) 9 h; (E) 12 h. Then total RNA were isolated.
The RT-PCR analysis using CCT and GAPDH primers was followed. n =
4, the ratio of 0 h was chosen as
control and set as 1.00, cP
< 0.01, bP
< 0.05 vs control.
Neurons were treated with control (lane
A), 10-7 mol/L AVP4-8
(lane B), 10-7 mol/L
AVP4-8+
5×10-6 mol/L ZDC(C)PR (lane
C), 5×10-6
mol/L ZDC(C)PR (lane D). n = 4,
aP < 0.05,
cP < 0.01 vs control, fP < 0.01 vs AVP4-8
(6 h).
Rat
hippocampal neurons were incubated in the presence of 5 mg/L actinomycin D and
10-7 mol/L AVP4-8
for 0, 1.5, 3, 4.5 and 6 h (the
control groups were treated without AVP4-8
). At the time indicated, total
RNA was isolated for RT-PCR and Southern analysis. As actinomycin D had no
effect on the turnover of GAPDH mRNA,
densitometric analysis of the CCT mRNA in the presence of actinomycinD
was normalized to the GAPDH mRNA. The results of the mRNA stability assay were
shown as decay curves (Fig.3). CCT mRNA was more stable in neurons coincubated
with AVP4-8, the statistic results of CCT/GAPDH
ratio for 0, 1.5, 3, 4.5, 6 h are
1.00±0.08, 1.01±0.08a, 0.89±0.05b, 0.87±0.05c, 0.77±0.09c respectively ( n = 3, the ratio of 0 h was set as 1.00, aP > 0.05, bP < 0.05, cP < 0.01 vs 0
h), the corresponding statistics of
control group are 1.00±0.07, 0.83±0.14a, 0.74±0.04b, 0.60±0.03c, 0.31±0.05c
( n = 3, the ratio of 0 h
was set as 1.00, aP
> 0.05, bP
< 0.05, cP
< 0.01 vs 0 h). This increased stability was correlated with the observed
increase in CCT mRNA.
(A) Treated with actinomycin D and AVP4-8;
(B) Treated with actinomycin D alone. The ratio of CCT and GAPDH at 0 h is considered
as control ratio and set as 1.00; n = 3, aP > 0.05, bP < 0.05, cP < 0.01 vs control. ○, actinomycin D; ●, actinomycin D +AVP4-8.
The
increase in CCT mRNA content was also accompanied by an increase in enzymatic
activity in cell lysates. Rat hippocampal neurons were incubated with AVP4-8
for 8 h, then CCT activity in
crude lysates was assayed. CCT activity was determined by measuring the rate of
incorporation of [14C]-phosphocholine into CDP-choline. CCT assays
were performed by the method of Lykidis et al.[20]. The enzyme
activity in AVP4-8
treated neurons increased for 86% according to untreated neurons, the relative activity for control and
AVP4-8
treated neurons are 1.00±0.10
and 1.86±0.17, ( P < 0.01 ) ( Fig.4).
a— d, neurons treated with AVP4-8
for 8 h; e-h, control. All incubations were performed
with 0.2 mCi
[14C]-phosphocholine and 50 mg
protein for 30 min at 37 ℃
in 50 ml
of total reaction volumn.
3
Discussion
In
the previous report, we found that
AVP4-8
could upregulate the CCT mRNA in rat hippocampus[13]. The mechanism
by which AVP4-8
upregulate CCT mRNA was investigated in this paper. To select an in vitro
system for the research, the
effect of AVP4-8
on CCT expression in primary rat hippocampal neuron was investigated. As not
enough RNA for Northern blot could be collected, RT-PCR analysis was introduced, to exclude the possibility of DNA contamination, PCR was performed without reverse
transcription and no band was observed. In our previous work we proved that AVP4-8
function most effectively on stimulating PKC in SK-N-SH cell and MAPK in
hippocampal neuron at 10-7 mol/L,
so we select this concentration for our research.
The
results demonstrated that CCT mRNA in rat hippocampal neurons could be
upregulated by AVP4-8, and the antagonist ZDC(C)PR could
inhibit this effect. After that,
the degradation rate of CCT mRNA in rat hippocampal neurons was
measured, sufficient actinomycin D
was added into the medium to inhibit mRNA synthesis in the neurons. The result
of RT-PCR and Southern analysis showed that CCT mRNA was more slowly degraded
in neurons coincubated with AVP4-8
than which simply treated with actinomycin D. This suggested that AVP4-8
could stabalize the CCT mRNA,
according to our recent finding that AVP4-8
could also stabalize the c-fos mRNA in rat hippocampal astrocytes (unpublished
result), this mechanism might be
one of the mechanisms by which AVP4-8
upregulate gene expression in rat brain. Yet we can not exclude the possibility
that AVP4-8
could also regulate the CCT mRNA at the transcriptional level.
Regulation
of CCT generally occurs at the enzyme level, such as through phosphorylation and lipid association, yet evidences for the existance of
pretranslational regulation accrued in these days, for example,
stimulation of quiescent cells with colony-stimulating factor causes a
4-fold increase in CCT mRNA levels by reducing the rate of RNA degradation[21], and in maturing typeⅡcells
there is a developmental increase in CCT mRNA caused by mRNA stabilization[22], yet the molecular mechanism for this
increase in mRNA stability remain to be investigated.
We
also verified that the increase of CCT mRNA induced by AVP4-8
caused CCT activity upregulation in rat hippocampal neurons. As phosphorylation
and lipid association usually lead to radical increase of CCT activity, we proposed that the regulation at mRNA
level might be one of the pathways which regulate CCT activity moderately.
Considering the function of PC and CDP-choline on memory and treatment of
AD, these results further
supported the previous suggestion that in rat hippocampus, some functions of AVP4-8
were at least partly performed through CCT-related pathway.
In
conclusion, the results in this
paper proved that AVP4-8
upregulated the CCT mRNA in rat hippocampal neurons by stabilizing it's
mRNA, and this upregulation
further led to the mild increase of CCT activity.
1 Liu RY, Lin C, Du YC.
Facilitation of arginine-vasopressin analogs on learning and memory in rats. Acta
Pharmacol Sin, 1990, 11: 97-100
2 Du YC, Yan QW, Qiao
LY. Function and molecular basis of action of vasopressin 4-8 and its analogues in rat
brain. Prog Brain Res,
1998, 119: 163-175
3 Du YC, Guo NN, Chen
ZF. Autoradiographic approach to the developmental study on the binding sites
of AVP4-8
in rat hippocampus. Acta Physiol Sin, 1994, 46: 435-440
4 Chen XF, Tang T, Zhang
JW, Miao HH, Wang TX, Du YC. ZNC(C)PR affects developmental changes of p46
phosphorylation in rat hippocampus. Mol Reprod Dev, 1993, 35: 251-256
5 Gu BX, Du YC. Arginine-vesopressin C-terminal peptide stimilates
inositol phospholipid metabolism in rat hippocampus. Acta Biochim Biophys
Sin, 1991, 23: 331-337
6 Dong M, Xu KY, Zhen
XG, Du YC. Arginine vasopressin(4-8) mobilizes intracellular
calcium in C6 glioma cells. Acta Biochim Biophys Sin, 2000, 32: 533-536
7 Zhen XG, Du YC. AVP(4-8) enhances PKC and MAPK activities in
SK-N-SH cells. Acta Biochim Biophys Sin, 2000, 32: 105-108
8 Dong M, Xiong Y, Xu KY, Du YC. ZNC(C)PR induces phosphorylation
of CREB in rat hippocampus. Acta Biochim Biophys Sin, 2000, 32: 575-580
9 Gu BX, Du YC. The neuropeptide ZNC(C)PR can induce c-fos and c-src
transcriptions in the hippocampus of newborn rats. Acta Biochim Biophys Sin, 1991, 23: 537-542
10 Zhou AW, Li WX, Guo
J, Du YC. Facilitation of AVP(4-8) on gene expression of BDNF
and NGF in rat brain. Peptides,
1997, 18: 1179-1187
11 Li WX, Gu BX, Du YC.
Effects of ZNC(C)PR and its analogs on CDNF mRNA expression in rat brain. Acta
Biochim Biophys Sin,
1991, 31: 249-253
12 He M, Chen XF, Du YC.
Effect of arginine-vasopressin short analogs on the growth of C6 cells. Chin
J Cell Biol, 1995, 17: 176-180
13 Xiong Y, Liu XL, Wang
Y, Du YC. Cloning of cytidine
triphosphate: phosphocholine
cytidylyltransferase mRNA upregulated by a neuropeptide arginine-vasopressin(4-8) in rat hippocampus. Neurosci
Lett, 2000, 283: 129-132
14 Billah MM, Anthes JC. The regulation and cellular functions of
phosphatidyl- choline hydrolysis. Biochem J, 1990, 269: 281-291
15 Exton JH. Signaling through
phosphatidylcholine breakdown. J Biol Chem, 1990, 265: 1-4
16 Cacabelos R, Caamano J,
Gomez MJ, Fernandez-Novoa
L, Franco-Maside A, Alvarez XA. Therapeutic effects of
CDP-choline in Alzheimer's disease,
Cognition, brain
mapping, cerebrovascular hemodynamics, and immune factors. Ann NY Acad Sci, 1996, 777: 399-403
17 Freshney IR. Culture of Animal Cells, a Manual of Basic Technique, 4th ed, New York:
Wiley-Liss Inc, 2000
18 Krumlauf R. Northern blot analysis.
In: Harwood A ed, Methods in Molecular Biology: Basic DNA and RNA protocols, Vol.58, Totowa: Human Press Inc,
1995, 113-128
19 Sambrook J, Fritish EF,
Maniatis T. Molecular Cloning:
A Laboratory Manual,
2nd ed, New York: Gold Spring Harbor Laboratory
Press, 1989
20 Lykidis A, Murti KG,
Jackowski S. Cloning and characterization of a second human CTP:
Phosphocholine cytidylyltransferase. J Biol Chem, 1998, 273:
14022-14029
21 Tessner TG, Rock CO, Kalmar
GB, Cornell RB, Jackowski S. Colony-stimulating factor
1 regulates CTP: Phosphocholine cytidylyltransferase mRNA levels. J Biol
Chem, 1991, 266: 16261-16264
22 Hogan M, Kuliszewski M, Lee W, Post M. Regulation of phosphatidylcholine synthesis in
maturing typeⅡcells: Increased mRNA stability of CTP:
Phosphocholine cytidylyltransferase. Biochem J, 1996, 314: 799-803
Received:
December 18, 2001
Accepted: January 14, 2002
This
work was supported by the Special Funds for Major State Basic Research Project(973) of China (No.G1999054000 )
*Corresponding
author: Tel, 86-21-64374430; Fax,
86-21-64338357；e-mail，
duyc@ sunm.shnc.ac.cn