PowerPointPrsentation

1
Protective function of Selenoprotein P in human
astrocytes
Lirija Alili , Holger Steinbrenner, Helmut Sies,
Peter Brenneisen
Institute for Biochemistry and Molecular Biology
I, Heinrich-Heine-University Duesseldorf, Germany
lirija.alili_at_uni-duesseldorf.de
INTRODUCTION
Immunostaining of SeP?Cells were grown on glass
coverslips and fixed with 3,7 formaldehyde
followed by 0,1 Triton X-100. After washing and
blocking, they were incubated with polyclonal
primary antibody against human SeP overnight at
4C. Secondary antibody used was Alexa fluor-488
labeled goat anti-rabbit IgG. Coverslips were
analyzed on a Zeiss Axiovert 100 TV inverted
microscope with 63x oil objective ( 20
µm).
Fig.6 Selenium supplementation increases the
expression and enzymatic activity of cytosolic
GPx
The essential trace element selenium is
incorporated into selenoproteins as
selenocysteine. Selenoprotein P (SeP) is the only
known selenoprotein with 2 selenocysteine-insertio
n sequence (SECIS) elements and multiple UGA
codons in its mRNA sequence, resulting in 10
selenocysteine residues, while all other known
selenoproteins carry only one selenocysteine 1.
SeP is the major selenoprotein in human blood
plasma 2, where its concentration has been
estimated to be 40 nM 3. Its expression was
detected in most tissues, and highest amounts are
produced by the liver, which secretes highly
glycosylated SeP with a molecular mass of 67 kDa
into plasma 4,5. The original hypothesis of SeP
as a transport protein, carrying selenium to
various extrahepatic tissues 6, was recently
supported by two studies demonstrating a
disturbed selenium distribution in SeP knock-out
mice 7,8. In addition, SeP has been shown to
chelate heavy metals such as cadmium and mercury
9, and there are several reports about its
antioxidant capacity It was shown to protect
from diquat-induced oxidative damage in rats 10
and to reduce phospholipid hydroperoxides in a
cell-free in-vitro system 11. Selenium is
involved in the maintenance of functional brain
activity and in protection against oxidative
stress-related brain disorders such as
Parkinson's disease 12. This idea is supported
by the finding that the brain retains high
selenium levels even under conditions of
persistent dietary selenium deficiency 13. SeP
is the principal supplier of selenium for the
brain 7,8 and was identified as a
survival-promoting factor for neuronal cells in
culture 14. However, recent studies with
knock-out mice demonstrated that, in the absence
of plasma SeP, other forms of selenium supply the
brain with this micronutrient, maintaining the
normal function of the central nervous system
8,15. Taken together, a summary of the effects
of selenium and SeP on human health is given by
Brenneisen et al. 16. In the present study, we
investigated the expression of SeP in human
astrocytes and its involvement in the protection
of these cells against oxidative damage.
MATERIAL AND METHODS
Subcellular fractionation of astrocyte
proteins?Fractions of cytosolic,
membrane/organelle and nuclear proteins of
MOG-G-CCM astrocytoma cells were prepared using
the ProteoExtract Subcellular Proteome Extraction
Kit (Merck Biosciences Bad Soden, Germany)
according to manufacturer's instructions.
Subsequently, the membrane/organelle fraction was
centrifuged with 20000 ? g for 30 min at 4C to
separate the mitochondria from the membranes.
si-RNA transfection?Specific downregulation of
SeP in MOG-G-CCM astrocytoma cells was done by
transfection with siRNA using X-tremeGENE siRNA
transfection reagent (Roche) according to
manufacturer's instructions. Cells were grown on
? 3,5 cm dishes until 70 confluency, and
transfected with 2,5 µl transfection reagent and
50 pmol SeP-specific siRNA (5'- GGA AUC UCU UCU
CGA UUA A -3' Qiagen) or unspecific control
siRNA (BLOCK-iT Invitrogen) in 1 ml OptiMEM
medium (Invitrogen). After 14 hours, transfection
medium was replaced with MOG-G-CCM medium, and 24
h upon transfection, SeP downregulation was
evaluated by immunoblotting. In a parallel set of
experiments, cell viability of siRNA-transfected
cells was determined by MTT assay.  
kDa 55 40 72 55 40 17 11
55
40
33
72
55
40
Fig. 1 Expression of Selenoprotein P in
MOG-G-CCM human astrocytoma cells (A) and in
human HepG2 liver cells (B)
SUMMARIZED RESULTS

• human astrocytes produce SeP as an unglycosylated
  intracellular protein, which is not secreted
• human astrocytes maintain a constitutive
  expression of SeP, which is independent of
  exogenous selenium supply
• Treatment with selenite, selenomethionine or
  exogenously added SeP protects human astrocytes
  from t-BHP-induced cytotoxicity
• in the protection of astrocytes against
  t-BHP-induced oxidative damage, human astrocytes
  use selenium independently of the selenium
  compound
• selenium supplementation results an cGPx mediated
  protection from t-BHP cytotoxicity
• selenium-deficient astrocytes with suppressed SeP
  expression are more sensitive to t-BHP- induced
  oxidative damage than non- or control-transfected
  cells

100
Selenium-mediated protection of astrocytes from
oxidative damage?MOG-G-CCM astrocytoma cells were
cultured in 24-well plates (Greiner). At 90
confluency, cells were grown in serum-free
medium, supplemented with 100 nM sodium selenite,
10 µM selenomethionine or 0.6 nM selenoprotein P
(from concentrated HepG2 supernatant) for 2 days.
Oxidative damage was caused by exposure to
tert.-butyl hydroperoxide (t-BHP) for 24 h. Cell
viability was determined by measurement of the
ability of the cells to metabolise MTT
(3-4,5-dimethyl-thiazol-2-yl-2,5-diphenyltetrazo
lium bromide) to a purple formazan dye as
described 17.
CONCLUSION
RT-PCRTotal RNA was prepared from MOG-G-CCM
astrocytoma cells by using RNeasyKit (Qiagen
Hilden, Germany) according to manufacturers
instructions. From each sample,1 µg of RNA was
transcribed into cDNA with SuperScript II reverse
transcriptase (Invitrogen) and p(dT)15 primers
(Roche Mannheim, Germany). PCR was performed
with Taq polymerase (Qiagen), using specific
primers. The forward primer for SeP was
5-CTTGGTTTGCCTTTTTCCTTCCTA-3 and the reverse
5-TTGCTGGCATATCTCGGTTCTCT-3. The PCR profile
was 3 min at 95C followed by 31 cycles of 30 sec
at 94C, 30 sec at 58C, and 1 min at 72C, as
well as a final elongation step for 5 min at 72C.
medium
The data suggest, that selenium-supplemented
human astrocytes are protected against
t-BHP-induced cytotoxicity by induction of
cytosolic GPx, while intracellular (endogenous)
SeP appears to have antioxidant activity under
selenium-deficient conditions.
125
100 nM sodium selenite
10 µM selenomethionine
100
0.6 nM SeP
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cell viability ()
75

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Fig. 2 Selenoprotein P mRNA expression in
MOG-G-CCM human astrocytoma is not altered by
selenium supplementation
50
25
0
0
50
100
200
tert.-butyl hydroperoxide (µM)
GPx activity assay?MOG-G-CCM astrocytoma cells in
? 10-cm dishes (Greiner) were cultured in
serum-free medium with or without selenium
supplementation for 2 d. Cells were lysed in GPx
assay buffer (100 mM Tris/HCl pH 7.6, 5 mM EDTA,
1 mM sodium azide, 0.1 Triton X-100), sonicated
and centrifuged with 5000 ? g for 10 min at 4C.
Enzymatic GPx activity in the cell extracts was
detected as described 19 using GPx assay buffer
supplemented with 3 mM glutathione, 600 mU/ml
glutathione reductase and 0.1 mM NADPH. Reaction
was started by addition of tert.-butylhydroperoxid
e to a final concentration of 50 µM and detected
by measurement of NADPH consumption at 340 nm
with a Lambda 25 spectrophotometer (Perkin
Elmer). GPx activity was determined using the
extinction coefficient of NADPH at 340 nm (?
6.2 mM-1cm-1).
Acknowledgement
This study was supported by Deutsche
Forschungsgemeinschaft, Bonn, Germany (SPP
Selenoproteine, Si 255/11-3). H. Sies is a
Fellow of the National Foundation for Cancer
Research (NFCR), Bethesda, MD.