Abstract
purpose. Maspin, a tumor-suppressor protein that regulates cell migration,
invasion, and adhesion, is synthesized by many normal epithelial cells,
but downregulated in invasive epithelial tumor cells. The purpose of
this study was to determine whether cells in the normal human cornea
express maspin and whether maspin affects corneal stromal cell adhesion
to extracellular matrix molecules.
methods. Maspin expression was analyzed by immunodot blot, Western blot, and
RT-PCR analyses in cells obtained directly from human corneas in situ.
Maspin protein and mRNA were also studied in primary and passaged
cultures of corneal stromal cells using Western blot analysis, RT-PCR,
and immunofluorescence microscopy. Maspin cDNA was cloned and sequenced
from human corneal epithelial cells and expressed in a yeast system.
The recombinant maspin was used to study attachment of cultured human
corneal stromal cells to extracellular matrices.
results. Maspin mRNA and micromolar amounts of the protein were found in all
three layers of the human cornea in situ, including the stroma. Maspin
was also detected in primary and first-passage corneal stromal cells,
but its expression was downregulated in subsequent passages.
Late-passage stromal cells, which did not produce maspin, responded to
exogenous recombinant maspin as measured by increased cell adhesion not
only to fibronectin, similar to mammary gland tumor epithelial cells,
but also to type I collagen, type IV collagen, and laminin.
conclusions. The corneal stromal cell is the first nonepithelial cell type shown to
synthesize maspin. Loss of maspin expression in late-passage corneal
stromal cells in culture and their biological response to exogenous
maspin suggests a role for maspin on the stromal cells in the cornea.
Maspin may function within the cornea to regulate cell adhesion to
extracellular matrix molecules and perhaps to regulate the migration of
activated fibroblasts during corneal stromal wound
healing.
Maspin is a member of the serine proteinase inhibitor
(SERPIN) superfamily of proteins that includes α-1 anti-trypsin,
plasminogen activator inhibitor, pigment epithelial–derived factor,
and ovalbumin.
1 Despite its similarity to other SERPINs,
it is unclear whether maspin functions as a proteinase
inhibitor.
2 3 Maspin is expressed by a variety of normal
epithelial cells of mammalian organs including mammary gland, prostate,
skin, stomach, and thymus.
4 5 6 This molecule is present
both within cells and associated with the extracellular matrix (ECM) of
these tissues. However, in mammalian carcinoma cell lines such as the
mammary gland epithelial tumor cell line MDA-MB-231, maspin expression
is downregulated, but the gene is not mutated.
7
Maspin is a tumor-suppressor protein that inhibits epithelial tumor
cell motility and invasion in vitro and suppresses tumor metastasis in
nude mice.
4 8 The inhibition of cell motility appears to
result from maspin’s ability to enhance tumor cell attachment to the
ECM molecule fibronectin.
9 Maspin is also an inhibitor
of angiogenesis, as indicated by its ability to inhibit bFGF-induced
proliferation and migration of microvascular endothelial cells in vitro
and to block neovascularization in vivo in the rat cornea pocket
model.
10 Therefore, maspin inhibits tumorigenesis, not
only by acting directly on the tumor cells, but also by inhibiting the
angiogenesis required for tumor growth.
We hypothesized that maspin is present in the cornea, a transparent
tissue which requires the absence of blood vessels and in which tumors
are rarely found.
11 As a product of many epithelial cell
types, we expected to find that maspin is synthesized in the corneal
epithelium and/or in the endothelium. In this study, these corneal
cells indeed synthesized maspin. Unexpectedly, maspin was also
expressed by the corneal stroma, which consists of nonepithelial cells
(keratocytes) surrounded by a largely collagenous matrix. Because
maspin is known to regulate the attachment of tumor cells to the ECM
molecule fibronectin, we considered that maspin may play a similar
regulatory role on ECM adhesion of corneal stromal cells during wound
healing. We studied corneal stromal cells, not only for their ability
to synthesize maspin, but also to determine whether stromal cells
treated with maspin exhibit altered adhesion to ECM molecules found in
normal or wounded corneas.
Human corneas (obtained from the Lions Eye Bank of Wisconsin)
were dissected and the epithelial, stromal, and endothelial layers
dissociated. Protein extracts from the tissues were prepared in 0.1 M
Tris-HCl buffer (pH 7.2) containing 0.15 M NaCl at 4°C. The
epithelial and endothelial layers were homogenized using a ground-glass
tissue grinder, and the stromal proteins extracted using a homogenizer
(Polytron; Brinkmann, Westbury, NY). To extract total RNA, the corneal
epithelial and endothelial layers were scraped and directly transferred
into extraction reagent (TRI Reagent; Molecular Research
Center, Cincinnati, OH). The stromal layer was ground in a percussion
chamber cooled with liquid nitrogen and then transferred into
extraction reagent. To prepare total RNA and total protein from the
cultured corneal stromal cells, extraction reagent was directly added
to the culture flasks. Isolation of total RNA and protein was then
performed as described in the manufacturer’s protocol.
Maspin cDNA was synthesized from 0.2 to 1 μg total RNA from
individual corneal layers or stromal cells using random hexamers and
murine leukemia virus (MuLV) reverse transcriptase (PE Biosystems,
Foster City, CA) at 42°C for 15 minutes and then amplified by the
polymerase chain reaction (PCR) using Taq DNA polymerase
(AmpliTaq Gold; PE Biosystems) according to the
manufacturer’s protocol. All oligonucleotide primers were from (Gibco
BRL-Life Technologies). A 468-bp PCR fragment was amplified using
primers specific to the reactive site loop (RSL) region of maspin
(5′-AGGATGTGGAGGATGAG-3′ and 5′-ACAGAAAAGTCAGGGAGG-3′). A three-step
temperature cycling for PCR was 1 minute at 95°C for denaturation, 1
minute at 55°C for annealing, and 1 minute at 72°C for extension. A
1.2-kb fragment containing the maspin open reading frame (ORF) was
generated using the PCR primers 5′-CGGAGATCTGCGGCCGCAATGGATGCCCTGC-3′
and 5′-CCGCTCGAGGAATTCA-CATGTGCTATGCCACT-3′. The PCR products were
cloned into a vector (pGEM-T Easy; Promega, Madison, WI), and then
manually sequenced (T7 Sequenase V 2.0 kit; Amersham Pharmacia
Biotech). The sequences were compared with the published human maspin
sequence from GenBank using the GCG software program (GenBank is
provide in the public domain by the National Center for Biotechnology,
Bethesda, MD, and is available at http//:www.ncbi.nlm.nih.gov/genbank).
Corneal stromal cells were cultured in eight chamber slides
(Nalge Nunc International, Rochester, NY). The cells were fixed for 15
minutes with cold 3% paraformaldehyde, then permeabilized by
incubation for 5 minutes at room temperature in 0.5% Triton X-100 in
phosphate-buffered saline (PBS). Fixed monolayers were incubated with
primary rabbit anti-maspin antibodies for 1 hour at room temperature,
followed by three rinses in blocking buffer (10 mg/ml BSA in PBS) and a
1-hour incubation in tetrarhodamine isothiocyanate
(TRITC)–conjugated donkey anti-rabbit IgG antibodies (Jackson
ImmunoResearch Laboratories Inc., West Grove, PA). Next, these
cells were costained for F-actin with FITC-phalloidin (Sigma) for 30
minutes at room temperature. After staining, coverslips were mounted
with antifade reagent (FluoroGuard; Bio-Rad), and specimens were
examined and photographed with a fluorescence microscope. Control
slides were stained using nonimmune rabbit IgG. All results were
confirmed in at least two independent experiments on samples from
different donors.
Because of the presence of an alpha factor leader sequence, the
recombinant protein was secreted into the yeast culture medium. The
cells were removed by centrifugation at 4500g, and the yeast
culture supernatant was concentrated to half the volume by
ultrafiltration, using a 30-kDa cutoff membrane (BioMax Millipore
Corp., Bedford, MA). The protein was buffer exchanged into a binding
buffer (50 mM phosphate buffer [(pH. 8.0]) containing 300 mM NaCl) by
diluting the sample 10× with buffer. The recombinant FLAG/His maspin
in the supernatant was purified using cobalt affinity resin (Talon
Superflow; Clontech, Palo Alto, CA). Large-scale batch purification was
performed according to the manufacturer’s protocol. The eluted
fractions containing recombinant maspin were pooled, and the imidazole
removed by repeated dilution with PBS and concentrated using a 30-kDa
cutoff centrifugal filter (Ultafree; Millipore).
The purified recombinant maspin was subjected to SDS-PAGE under
reducing conditions and detected by Coomassie blue staining
(Fig. 1B) or after electroblot transfer to nitrocellulose and then detected by
the nickel-conjugated HRP (INDIA HisProbe-HRP; Pierce;
Fig. 1B ). The
hexahistidine tag, the FLAG peptide composed of eight amino acids, and
glycosylation account for the extra 8-kDa over the native size of 42
kDa. Approximately 1 mg purified yeast recombinant maspin was produced
per liter of yeast culture medium.
The biologic activity of recombinant maspin was tested on mammary gland
carcinoma MDA-MB-231 cells using a cell–fibronectin adhesion assay, as
described in the following section. The recombinant protein was
biologically active, in that it increased the tumor cell attachment to
fibronectin, similar to the recombinant bacterial maspin obtained from
Phillip A. Pemberton (data not shown).
Cell adhesion assays were conducted using ECM-coated plates
(CytoMatrix; Chemicon International, Temecula, CA) according to the
manufacturer’s instructions.
MDA-MB-231 cells and corneal stromal cells were cultured in MEM plus
2% lactalbumin (Sigma) and the human stromal culture medium without
FBS, respectively. Subconfluent cells were pretreated overnight with 1μ
M yeast recombinant maspin. As a negative control, ovalbumin (grade
VII, essentially free of S-ovalbumin; Sigma), a SERPIN very homologous
to maspin, was included in the assay. The cells were harvested using
enzyme-free, Hanks’-based, cell dissociation buffer (Life
Technologies). They were washed with PBS, resuspended in medium, and
counted using a hemocytometer. Approximately 2 ×
104 cells were plated on cell adhesion strips
(CytoMatrix; Chemicon International), precoated with ECM proteins or
with BSA as a negative control. After incubation at 37°C for 1 hour,
the nonadherent cells were removed by gently washing with PBS
containing 1 mg/ml CaCl2 and 1 mg/ml
MgCl2. The adherent cells were stained with 0.2%
crystal violet in 10% ethanol. The excess stain was removed by gently
washing three times with PBS. The attached cells were then solubilized
with a 1:1 mixture of 0.1 M
NaH2PO4 (pH 4.5) and 50%
ethanol. Cell attachment was determined by measurement of dye color at
550 nm on an ELISA microplate reader. Each experiment contained at
least triplicate samples for each condition. Four independent
experiments were performed with cells from different donors.
Statistical analysis was performed (Sigma Stat software; SPSS Inc.,
Chicago, IL) using a one-way analysis of variance for overall
differences among control, maspin, and ovalbumin treatment groups. The
significance of differences between mean values of optical density at
550 nm was determined using the Student-Newman-Keuls method, with P < 0.05 indicating significance.