Pokaż cały numer - FPN - Farmaceutyczny PrzeglÄ d Naukowy

copyright © 2009 Grupa dr. A. R. Kwiecińskiego ISSN 1425-5073
phenomenon may be evoked by a decrease of collagen biosynthesis,
increased degradation of this protein or by coexistence
of both phenomena.
Obviously collagen biosynthesis requires energy supply.
Glucose is the main energetic substrate and glycolysis is the main
process which supplies energy for fibroblasts grown in vitro [5].
For these reasons the glucose shortage may reduce intracellular
pool of ATP required for the functions of these cells, including
collagen biosynthesis. Furthermore, the shortage of glucose in
culture medium enhances proteolytic (and gelatinolytic) activity
in these cells [1,6] and may promote collagen degradation.
On the other hand glucose deprivation appeared to be a
factor which induces the expression of oxygen-regulated protein
(ORP) 150 in fibroblast cultures [4]. Oxygen-regulated
protein of molecular weight 150 kDa (ORP150) and glucose
- regulated protein of molecular weight 170 (GRP170) are
endoplasmic reticulum (ER) chaperones, which facilitate
protein folding [2]. The GRP170 is a glycosylated form of
ORP150. The ORP150/GRP170 system is a part of the ER
machinery that assists in the folding and assembly of secretory
and membrane proteins within the ER [3]. The expression
of ORP150 increases in a range of pathologic situations
such as brain ischaemia [7], atherosclerotic plaques [8] and malignant
tumours [9-11] suggesting that ORP150 is a contributory
factor for the cellular response to environmental stress.
The role of ORP150 in cellular physiology remains unclear.
Some observations indicate that ORP150, like GRP78 and
GRP94, is involved in the processing of proteins in the secretory
pathway [12]. This allows suggesting that ORP150 is a chaperon,
which protects intracellular collagen against proteolytic effects
exerted by glucose shortage. In order to test such a hypothesis
we decided to study the effect of glucose deprivation in culture
medium on proline incorporation into total proteins and specifically
into collagenase-sensitive and hydroxyproline-containing
proteins. Furthermore, the degradation of newly synthesised
collagen and correlation of this process with the expression of
glucose-regulated proteins (ORP150/GRP170) was studied.
Materials and methods
Reagents
The DMEM was provided by Invitrogen (San Diego,
USA). Passive lysis buffer (Promega, Madison, USA), monoclonal
anti-human ORP 150 (IBL, Gunma, Japan), highly
purified bacterial collagenase (type VII), Sigma-Fast BCIP/
NBT reagent, alkaline phosphatase-labelled anti-mouse
immunoglobulin G were purchased from Sigma (St Louis,
USA), as were most other chemicals and buffers used.
[2,3,4,5 – 3 H] L-proline was obtained from Hartmann Analytic
(Braunschweig, Germany). Nitrocellulose membranes
(0.2 μm), Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) molecular weight standards
and Coomassie Brilliant Blue R-250 were purchased from
Bio-Rad Laboratories (Hercules, CA).
Cell cultures
The experiments were performed on the human skin
fibroblast cell line (CRL-1474) purchased from American
Type Culture Collection (ATCC). The cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM), containing
glucose at 4.5 mg/ml (high glucose DMEM) supplemented
with 10% heat-inactivated foetal calf serum (FCS), 2 mM
L-glutamine, penicillin (100 U/ml) and streptomycin (100
μg/ml). The cells were seeded at a density of 5 x 10 5 cells
in 2 ml medium and grown on six-well plates, in a 5 % CO 2
incubator, at 37 O C.
Experimental procedure
5 x 10 5 cells in 2 ml medium were seeded in six-well plates
and incubated for seven days in the high glucose DMEM. In
these conditions the cultured cells reached 70-80% confluency.
After this time the medium was removed and replaced
with 2 ml of the fresh DMEM (without calf serum):
1. the high glucose DMEM contained 4.5 mg of glucose
per ml (25mM),
2. the low glucose DMEM contained 0.5 mg of glucose per
ml (2.8 mM).
Each was supplemented with [2,3,4,5– 3 H] L-proline,
ascorbate (50 µg/ml), 2 mM L-glutamine, penicillin (100
U/ml) and streptomycin (100 μg/ml). The incubation was
continued for 12, 24, 48 hours. The fibroblast did not divide
during the incubation in serum-free medium. No increase in
cell density was observed in this period.
After incubation the culture media were removed, the cell
layers were washed with PBS and submitted to the action of
lysis buffer. It allowed separating the cells and extracellular
matrix from the bottom of culture vessels and suspending
them in the buffer.
Filtration Assay
Each hydrophilic Durapore membrane (0.65-µm pore
size) was soaked with 250µl of 25% TCA. The cell lysates
and culture media were treated with 50% TCA (final TCA
concentration 25%) and next incubated at 4 o C for 1h. The
formed precipitate was collected on the filter membranes
and separated from the supernatant using a vacuum source
attached to the 1225 Sampling Manifold (Millipore). To
remove all unincorporated [ 3 H]-proline, the filters were
washed 3 times with 5 ml of 10% TCA by vacuum-filtration
through the membrane. After removing the underdrain and
drying, the membranes were then punched out into scintillation
vials and the filter-bound radioactivity was quantified
by liquid scintillation counting [13,14].
The assay of collagenase-sensitive protein
The collagenase-sensitive protein was measured using
the method described by Petrkofsky and Diegelman [15].
The cell lysates and culture media were treated with high purity
bacterial collagenases (type VII-Sigma). N-ethylmaleimide
was applied as an inhibitor of unspecific proteolytic
activity associated with collagenase. The amount of newly
synthesised collagen was expressed in dpm of [ 3 H]-proline,
incorporated into protein susceptible to the action of bacterial
collagenases [15].
Determination of radioactive hydroxyproline
Cell lysate or incubation medium was submitted to hydrolysis
in 6 M HCl, at 100 O C, for 16 h. The hydrolysates
were evaporated to dryness in a rotary glass evaporator. The
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