(Investigative Ophthalmology and Visual Science. 2000;41:2707-2711.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
Synthesis of Osteonectin by Human Retinal Pigment Epithelial Cells is Modulated by Cell Density
Raymond M. Magee1,
Suzanne Hagan1,
Paul S. Hiscott1,2,
Carl M. Sheridan1,
John A. Carron3,
Jim McGalliard1 and
Ian Grierson1
1 From the Unit of Ophthalmology, Department of Medicine,
2 Department of Pathology, and
3 Department of Human Anatomy and Cell Biology, University of Liverpool, United Kingdom.
 |
Abstract
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PURPOSE. To determine whether human retinal pigment epithelial (HRPE) cells are
able to synthesize the antiadhesive protein osteonectin, also known as
secreted protein, acidic and rich in cysteine (SPARC). Additionally,
because locally produced SPARC may modulate cellular behavior during
tissue repair, to ascertain whether HRPE SPARC production and HRPE
proliferation, migration, and/or differentiation are associated, in a
simple HRPE wound-healing model.
METHODS. Immunohistochemical and Western blot analyses of SPARC protein
expression by low- and high-density cultured HRPE cells were
undertaken. Total RNA extracted from cultures was studied by reverse
transcription–polymerase chain reaction (RT-PCR) and Northern blot
analysis. Western and Northern blot analyses were evaluated by
densitometry. Experiments were repeated with HRPE cells cultured in the
presence of 1, 10, or 100 µM of the differentiating agents butyric
acid (BA) and retinoic acid (RA).
RESULTS. HRPE cell cultures exhibited SPARC immunoreactivity. Western blot
analysis of cell lysates and conditioned media showed a 43-kDa protein.
RT-PCR and Northern blot analysis confirmed the presence of SPARC mRNA
(with transcripts at 2.2 and 3.0 kb). Protein and mRNA transcript band
densitometry revealed a higher proportion of SPARC protein and mRNA in
high-density HRPE cell culture than in low-density culture. Neither BA
nor RA (at the concentrations assessed) had a significant effect on
SPARC production by HRPE cells in high- or low-density culture.
CONCLUSIONS. HRPE can synthesize SPARC. Although the findings do not support an
invariable association between SPARC production by HRPE and HRPE
proliferation, migration, or differentiation, they demonstrate that
synthesis of SPARC by HRPE is modulated by cell
density.
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Introduction
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The 43-kDa glycoprotein, secreted protein acidic and rich in
cysteine (SPARC), also known as osteonectin, is one of a group of
secreted extracellular matrix proteins (matricellular proteins) that
interact both with cell surfaces and a range of extracellular molecules
(e.g., growth factors) but that generally have no structural
role.1
Although the precise function of SPARC remains
unclear, it possesses counteradhesive properties that may permit
changes in cell shape necessary for such cellular activities as
proliferation and migration.2
Cell proliferation and
migration are features of development and repair and, indeed, SPARC is
produced in many developing organs and in healing
wounds.1
2
3
Conversely, SPARC is also produced by cells in
organs undergoing terminal differentiation, when cell proliferation and
migration may be decreasing. Thus, for example, SPARC increases through
differentiation in the chick retinal pigment epithelium and persists in
the avian adult monolayer.4
We have noted an association between human retinal pigment
epithelial (HRPE) cells and SPARC in the membranes of the anomalous
wound-repair condition proliferative vitreoretinopathy
(PVR),5
an observation that is consistent with the notion
that HRPE have the ability to synthesize SPARC. Moreover, because PVR
membranes contain migratory and proliferating cells on the one hand and
on the other hand cells in various stages of
transdifferentiation,6
it is possible that SPARC may be
produced by HRPE cells during proliferation, migration, and/or
differentiation. To investigate these possibilities, initially we
determined whether HRPE cells have the ability to synthesize SPARC.
Then we studied SPARC production in a simple in vitro model of wound
repair using HRPE in low-density cultures, where cells are isolated,
mitotic, and motile (i.e., in wound-repair phenotype), and in
high-density cultures where cells are in contact and proliferation and
migration are less marked. Finally, we investigated SPARC synthesis by
HRPE cells treated with retinoic acid (RA) or butyric acid (BA), agents
known to induce differentiation in these cells.7
8
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Materials and Methods
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HRPE and CRL2070 Cell Culture
HRPE cells were cultured in Ham’s F10 (Life Technologies,
Paisley, UK) containing 20% fetal calf serum (FCS; Harlan Sera
Laboratory, Crawley Down, UK), 1% each glutamine, fungizone (Life
Technologies), and 1% each glucose, penicillin-streptomycin (Sigma,
Gillingham, NY). Cultures were maintained in 5% CO2 at
37°C and grown to confluence in 150-cm2 flasks (Corning,
Corning, NY) before passaging. Purity of cell cultures was confirmed on
the basis of cytokeratin staining.9
Cultures were used in
experiments after the fourth passage. For experiments involving protein
and mRNA analyses, HRPE cells were seeded at 2 x 106
cells/flask using 75-cm3 and 182-cm3 flasks
(Griener Ltd., Frickenhausen, Germany). Cells were cultured for 4 days,
at the end of which the cells were either still of low-density
(preconfluent; 182-cm3 flasks) or high density (confluent;
75-cm3 flasks). The mouse teratogen SPARC-producing cell
line, CRL2070 (American Type Culture Collection, Rockville, MD) was
cultured in DMEM (Life Technologies) supplemented with 10% FCS and 1%
each of glutamine, fungizone, and penicillin-streptomycin. This cell
line provided controls for the Western and Northern blot analyses.
SPARC Staining of RPE In Vitro
HRPE cells were seeded at a density of 7.5 x
103 cells per well on eight-chamber tissue
culture slides (LabTek; Nunc, Naperville, IL) for immunofluorescence
staining, as described.9
Previously characterized
antiserum and monoclonal antibody to SPARC (LF-BON-1 and AON-1
respectively) were used.10
Cells were washed with
phosphate-buffered saline (PBS) and fixed in methanol (5 minutes) and
acetone (2 minutes) at -20°C. Nonspecific binding was blocked with
normal goat serum before incubating the cells with primary antibodies
at previously optimized dilutions of 1:1000 (LF-BON-1), 1:250 (AON-1)
in PBS10
(Developmental Studies Hybridoma Bank, University
of Iowa, Iowa City, IA). After a PBS wash, the RPE preparations were
treated with anti-rabbit or anti-mouse fluorescein isothiocyanate
(FITC) conjugate (1:100; Sigma). RPE monolayers were examined with a
microscope (Polyvar; Reichert-Jung, Germany) equipped with
epifluorescent optics.
Western Blot Analysis
Western blot experiments were conducted on cell lysates and
conditioned media from HRPE cells. The tumor cell line CRL2070,
together with pure SPARC protein (Hematologic Technologies, Essex
Junction, VT), were used as positive controls. Electrophoresis was
performed with 10% polyacrylamide gels. The bicinchoninic acid assay
(Sigma), was used for the determination of protein content in the
samples before electrophoresis. Cell lysates were dissolved in sample
buffer containing ß-mercaptoethanol before loading on the gels. For
subsequent immunostaining, the proteins were transferred by
electrophoresis to nitrocellulose membrane (Sigma). Unspecific binding
to the membranes was blocked using a 10% dried milk in TRIS-buffered
saline (TBS; 20 mM TRIS and 137 mM sodium chloride [pH7.6]; Sigma)
containing 0.1% Tween-20 (Sigma) at 4°C overnight. Membranes were
incubated for 1.5 hours at room temperature with the monoclonal
antibody AON-5031 (Hematologic Technologies) at a dilution of 1:5000.
Bound antibody was visualized using a goat anti-mouse horseradish
peroxidase–conjugated secondary antibody (Sigma), at a dilution of
1:2500 (containing 25% new born calf serum) for 1 hour and subsequent
chemiluminescent reaction using a commercial system (ECL; Amersham,
Amersham, UK). Each experiment was conducted three times.
RNA Extraction and cDNA Synthesis
For each experiment, RNA was extracted from RPE monolayers using a
kit (RNEasy; Qiagen, Crawley, UK). RNA (5 µg) was used as template
for first-strand cDNA synthesis in a 25-µl reaction volume containing
5 µl 5x first-strand buffer (Life Technologies); 0.5 mM each of
dATP, dCTP, dGTP, and dTTP; 1.25 µg oligo(dT) (Pharmacia, St. Albans,
UK); 20 U RNase inhibitor; 10 mM dithiothreitol; and 2 µl reverse
transcriptase (Superscript; Life Technologies). The reaction was
incubated at 37°C for 1 hour and terminated by freezing.
Reverse Transcription–Polymerase Chain Reaction
Reverse transcription–polymerase chain reaction (RT-PCR) was
performed in a 50-µl reaction mixture containing the following
reagents: 0.25 µl DNA polymerase (Thermoprime; Advanced
Biotechnologies, Epsom, Surrey, UK); 5 µl 10x PCR buffer; forward
and reverse primers (0.5 µl of 1 µg/µl); 200 mM each of dATP,
dCTP, dGTP and dTTP; 1.5 mM MgCl2; and 2 µl
cDNA preparation. The cycling conditions for all primers were as
follows: stage 1, 94°C for 2 minutes; stage 2, 35 cycles of 94°C
for 30 seconds, 57°C for 30 seconds, 72°C for 60 seconds; and stage
3, 72°C for 5 minutes. The primers used for PCR were as follows:
SPARC 5'-GATGCGCTGACCACTTC-3'; 5'-CGCATTAATAGTCCCATTTTT-3'; GAPDH
5'-GGTCAGGAGTCCCTTCCACGAT-3'; and
5'-GGTGAAGGTCGGATGTCAACGG-3'. PCR products were separated by
electrophoresis on a 1% agarose gel and the products visualized under
ultraviolet illumination.
Northern Blot Analysis
Total cellular RNA was extracted using a kit (RNeasy; Qiagen).
Total RNA (5 µg/lane) was denatured with formaldehyde (Sigma) and
formamide (Sigma) and was size fractionated by electrophoresis through
a 1% agarose gel (Life Technologies) containing formaldehyde. The RNA
was then transferred to a nylon membrane (ICN, Irvine, CA) overnight
and cross-linked with ultraviolet irradiation. A cocktail of four
SPARC-specific, 3'-tailed, digoxigenin (DIG)-labeled oligonucleotides
(Life Technologies) was used to probe the blotted membrane. Even
loading of RNA samples was assessed through use of a 5' and 3'
DIG-labeled 18S ribosomal RNA probe (Euro-Gentec, Oxford, UK). The
blotted membrane was hybridized overnight at 42°C (Easy Hyb;
Boehringer Mannheim, Indianapolis, IN). The membrane was successively
washed at room temperature with 2x SSC (three times for 5 minutes; one
time for 10 minutes), at 48°C with 0.1x SSC (two times for 15
minutes), and at room temperature with maleic acid washing buffer (0.1
M maleic acid, 0.15 M sodium chloride, and 0.3% Tween 20; one time for
3 minutes). Bands were visualized using a commercial system (CDP-Star;
Boehringer Mannheim), and the blot was exposed to film (Hyperfilm;
Amersham). The 3'-tailing of SPARC probes was performed using a DIG
oligonucleotide 3'-tailing kit (Boehringer Mannheim). Each experiment
was conducted three times.
Effect of BA and RA on SPARC mRNA Expression
For all experiments fresh BA and RA were used. Experiments were
conducted under darkened conditions. Preconfluent and confluent HRPE
cells were exposed to 1 µM, 10 µM, and 100 µM BA (sodium salt;
Sigma) and 1 µM, 10 µM, and 100 µM RA (Sigma) in Ham’s F10
medium supplemented with 10% FCS for 24 hours and 72 hours,
respectively. The preparations were incubated at 37°C and 5%
CO2. After each experiment, protein and total RNA
were extracted from cells, subjected to Western and Northern blot
analyses, respectively, followed by densitometric analysis. Experiments
were conducted a minimum of three times.
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Results
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SPARC Protein and HRPE Cells
RPE cells were immunoreactive for SPARC, and a similar pattern of
staining for the protein was observed in both preconfluent and
confluent HRPE cell cultures. The pattern of SPARC immunostaining in
HRPE cells was of a granular perinuclear and a diffuse peripheral
punctate nature (Fig. 1)
. Immunostaining associated with the extracellular matrix was also
observed and appeared to consist of discrete foci of SPARC
immunopositivity arranged in threadlike or beadlike patterns (Fig. 1)
.
Positive immunostaining was observed with both SPARC antibodies
(LFBON-1 and AON-1). Controls in which the primary antibodies were
omitted showed no immunoreactivity (Fig. 1)
.
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Figure 1. (A) Immunohistochemical localization of SPARC in an HRPE
cells monolayer using antiserum LF-BON-1. Similar staining was observed
using monoclonal antibody AON-1. Arrowheads: perinuclear
staining; arrows: discrete foci of SPARC immunostaining
associated with the extracellular matrix. (B) Negative
control in which the primary antibody was omitted from the staining
procedure. Bar, 10 µm.
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Immunoblot of HRPE cell lysates and conditioned medium (standardized
for protein concentration at 50 µg/lane) demonstrated the presence of
a 43-kDa band (the expected molecular weight of SPARC) that displayed
immunoreactivity with the monoclonal antibody AON-5031 (Fig. 2)
. A band of the same molecular weight was observed in CRL2070 cell
lysates and with purified SPARC protein. Densitometric analysis of the
bands representing SPARC from cultured HRPE cells (conducted on
standardized amounts of total protein and at the same time interval in
culture) revealed significantly higher concentrations (14-fold
increase; P < 0.05; t-test) of SPARC in
confluent than in preconfluent cultures (Fig. 2)
.
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Figure 2. Western blot analysis of cell lysates (equal protein loaded at 50
µg/lane) from preconfluent (lane 1) and confluent HRPE
cells (lane 2). Also shown is cell lysate from the
SPARC-producing cell line CRL2070 (lane 3).
Arrow: 43-kDa protein, the expected molecular size of
SPARC.
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SPARC mRNA and HRPE Cells
The primers designed to detect cDNA for SPARC produced a band of
predicted size (514 bp), with the use of control cDNA from the CRL2070
SPARC-producing cell line (data not shown). When these primers were
used on cDNA from cultured HRPE cells, SPARC mRNA was detected in
preconfluent and confluent cells (Fig. 3) . PCR using primers for the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
demonstrated the integrity of the cDNA from the cultured HRPE cells.
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Figure 3. (A) RT-PCR of human SPARC in HRPE cells from preconfluent
(lane 1) and confluent (lane 2) cultures. A
514-bp band consistent with SPARC mRNA was identified in each reaction.
The 500-bp band represents the GAPDH reaction control. (B)
Northern blot analysis showing expression of SPARC mRNA in total RNA
extracts from preconfluent HRPE cells (lane 2), confluent
HRPE cells (lane 3), and the CRL2070 SPARC-producing cell
line (lane 4). Lane 1: DIG-labeled molecular
weight markers.
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Northern blot analysis of total RNA from cultured HRPE cells identified
two mRNA transcripts migrating at 2.2 and 3.0 kb (Fig. 3)
equivalent to
that expressed by other human cells. The intensity of the 3.0-kb band
appeared less than that of the 2.2-kb band. Only a single band of 2.2
kb was observed for the CRL2070 SPARC-producing cell line, which was
consistent with its murine origin.2
Densitometric analysis
of the transcript bands from cultured RPE (conducted on standardized
amounts of total RNA and at the same time interval in culture) revealed
significantly higher levels of SPARC mRNA expression (2.5-fold
increase; P < 0.01; t-test) in confluent
than in preconfluent cultures (Fig. 3)
.
Effect of BA and RA on SPARC mRNA Expression
HRPE cells were exposed to RA and BA for times previously
established to induce features associated with HRPE cell
differentiation7
and to induce SPARC synthesis in other
cell types.11
12
All three concentrations of RA induced
features that have been described previously, including cellular
flattening and polygonal shape.8
Similar changes were also
observed in all the BA-treated cells, although these changes were not
so pronounced as in the RA-treated cells. Treatment of preconfluent and
confluent HRPE cells with 1 µM, 10 µM, or 100 µM BA or 1 µM, 10
µM, and 100 µM RA for their respective incubation periods, induced
no significant increase in SPARC protein or in mRNA expression (Fig. 4
; data shown for BA only).
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Figure 4. Northern blot analysis showing expression of SPARC mRNA in total RNA
extracts from preconfluent and confluent HRPE cells before exposure of
1 µM BA (lanes 2 and 4, respectively)
and after exposure to 1 µM BA (lanes 3
and 5, respectively). Lane 1 represents
the DIG-labeled molecular markers.
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Discussion
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Our results demonstrate that HRPE cells are capable of
synthesizing SPARC. Moreover, some of the protein appears to be
secreted by the cells and deposited in the extracellular matrix,
consistent with a proposed role for SPARC in extracellular matrix
assembly.2
In addition, we have shown that the proportion
of SPARC to total protein in high-density cultures is greater than in
low-density cultures and that this difference in SPARC expression is
paralleled by higher SPARC mRNA levels in confluent HRPE cells. These
observations suggest that the higher SPARC level in confluent cultures
is, at least in part, due to increased production of the protein rather
than decreased SPARC degradation at high cell density.
SPARC is produced in tissues undergoing development and repair. We have
previously shown that SPARC is colocalized with HRPE cells in the
anomalous reparative tissue of PVR membranes.5
Given
possible roles for SPARC in repair-related cellular activities such as
proliferation, migration, and differentiation, we questioned whether
RPE-derived SPARC might be linked to HRPE cell proliferation,
migration, and/or differentiation.
To investigate the possibility that SPARC production by HRPE cells is
associated with differentiation of the cells, we used a range of
concentrations of two established differentiating agents with both
high- and low-density HRPE cell cultures. Although, as previously
reported, the RA- and BA-treated HRPE cells adopted features similar to
mature cells in vivo,7
8
and the difference in SPARC
synthesis between low- and high-density culture was maintained
irrespective of treatment, neither RA nor BA elicited any significant
change in SPARC production by HRPE cells. BA and RA upregulate SPARC
synthesis by some but not all cells. For example, RA increases SPARC
expression by chick chondrocytes11
but has little effect
on SPARC synthesis by human osteoblasts.13
Variance
between cells in the effect of differentiating agents on SPARC
production may reflect either species or cell type differences, or
both. However, although BA and RA induce many of the features typical
of differentiated HRPE cells (such as polygonal shape and arrested
growth and migration),7
8
these agents do not appear to
produce a mosaic of polarized HRPE cells (i.e., tertiary
differentiation) in vitro.6
Thus, we cannot exclude the
possibility that the cell density–dependent increase in HRPE SPARC
synthesis is related to events at the end of retinal development.
A number of cell types exhibit increased SPARC synthesis when cell
proliferation is in decline.1
2
Our observation that SPARC
expression is upregulated in high-density culture (where HRPE cell
proliferation is generally less abundant than in low-density culture)
is consistent with the notion that increased SPARC synthesis by HRPE
cells is associated with a decrease in HRPE cell proliferation.
However, the finding that neither RA nor BA (which both inhibit RPE
proliferation)7
8
increase HRPE SPARC synthesis suggests
that termination of HRPE cell proliferation can occur without any need
for increased SPARC production by these cells. Furthermore, because
HRPE cell migration also is decreased in high-density cell culture, our
findings do not support the concept that HRPE cell migration is related
to increased SPARC synthesis by the cells, although further
studies are required to establish the relationship between HRPE cell
migration and SPARC synthesis.
The findings of our study suggest roles for HRPE-derived SPARC
other than, or in addition to, HRPE cell proliferation, migration, and
differentiation. SPARC has been implicated in the regulation of
angiogenesis.1
It is well established that HRPE cells have
an important function in the control of angiogenesis at the
chorioretinal interface and that this function is mediated by a variety
of HRPE-derived peptides and proteins (see, for example, recent review
by Campochiaro14
). Therefore, it is possible that
production of SPARC by HRPE cells plays a role in the biology and
pathobiology of vascularization at this site. If this is the case, we
could expect an involvement of SPARC in the vascular abnormalities
associated with age-related macular degeneration. We are currently
investigating this concept.
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Acknowledgements
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The authors thank Larry Fisher who kindly supplied the LF-BON-1
antibody.
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Footnotes
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Supported by The Guide Dogs For The Blind Association; The Royal College of Surgeons of Edinburgh and The Blind Asylum; National Health Service Executive North West Regional Research and Development Directorate; The University of Liverpool Research Fund; Research into Eye Disease; and the Foundation for the Prevention of Blindness.
Submitted for publication November 17, 1999; revised March 20, 2000; accepted April 11, 2000.
Commercial relationships policy: N.
Corresponding author: Paul Hiscott, Unit of Ophthalmology, Department of Medicine, University Clinical Departments, Duncan Building, Liverpool L69 3GA, UK. p.s.hiscott{at}liv.ac.uk
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References
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Abstract
Introduction
