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1 From the Departments of Biochemistry and 2 Ophthalmology, Medical College of Wisconsin, Milwaukee.
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Abstract |
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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.
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Introduction |
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-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.
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Methods |
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Human corneal stromal cells were recovered by collagenase digestion of human corneas obtained from the Wisconsin Lion Eye Bank (Milwaukee, WI), as previously described by Taylor et al.12 The cultured corneal stromal cells were grown in flasks (Costar, Cambridge, MA) containing high-glucose DMEM with L-glutamine (Life Technologies) supplemented with 5% FBS defined (Hyclone, Logan, UT), 0.1% Mito+ serum extender (Collaborative Research, Bedford, MA), and 10 µg/ml ciprofloxacin (Bayer, Kankakee, IL) at 34°C. Cells were split at a ratio of 1:2 or 1:3.
Preparation of Protein Extract and Total RNA
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.
Western Blot Analysis
Supernatant fractions of each extract were electrophoresed on
10% SDS-PAGE gels under reducing conditions, and electrotransferred to
nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ),
as described elsewhere.13
Maspin was detected in the
epithelial and endothelial extracts by rabbit anti-human maspin IgG
antibodies affinity purified on a maspin column and shown to be
specific for maspin.6
The maspin antibodies were generous
gifts from Phillip A. Pemberton (LXR Biotechnology, Richmond, CA).
Nonspecific rabbit IgG (Bio-Rad, Hercules, CA) was used as a control on
duplicate blots. Affinity-purified goat anti-rabbit IgG conjugated with
horseradish peroxidase (HRP; Bio-Rad) was used as the secondary
antibody. For the stromal extract, a mouse monoclonal anti-human maspin
antibody (BD PharMingen, San Diego, CA) was used as a primary antibody
and HRP-conjugated goat anti-mouse IgG (Pierce, Rockford, IL) as the
secondary antibody. To detect corneal epithelium–specific cytokeratin
3, a mouse monoclonal anti-human cytokeratin-3 antibody (Research
Diagnostics, Inc., Flanders, NJ) was used as the primary antibody and
goat anti-mouse IgG conjugated with HRP (Pierce) as the secondary
antibody. For detection of recombinant FLAG/His-tagged maspin, a
nickel-conjugated HRP (INDIA HisProbe-HRP; Pierce) was used. The
HRP-labeled bands were visualized using an enhanced chemiluminescence
(ECL) detection system (Amersham Pharmacia Biotech). Unless otherwise
indicated, all results were confirmed in at least two independent
experiments on tissue extracts or cultured cells from different donors.
Immunodot Blot Assay
The total amount of protein extracted from the corneal
epithelial, stromal, and endothelial layers was determined by the Lowry
assay using bovine serum albumin (BSA; Sigma) as a standard
protein.14
The supernatant fraction of corneal total
protein extracts and bacterial recombinant maspin standards were loaded
onto a nitrocellulose membrane in a 96-well dot blot apparatus
(Schleicher & Schuell, Keene, NH). The dot blot was processed in the
same manner as described for the Western blot analysis, using rabbit
polyclonal anti-human maspin antibodies for the epithelial and
endothelial extracts and the mouse monoclonal anti-human maspin
antibody for the stromal extracts. Nonspecific binding was determined
on duplicate blots using nonspecific rabbit or mouse IgG plus the
secondary antibodies or the secondary antibodies alone as described for
the Western blot analysis. The data were analyzed using an ELISA plate
reader (EL380 Microplate Reader; Bio-Tek Instruments, Winooski, VT)
with a 530-nm filter. Each sample was assayed in triplicate. Linear
regression analyses of standard maspin preparations were used to
determine the maspin concentration in each sample. The values were
averaged and the SD was determined.
Reverse Transcription–Polymerase Chain Reaction
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).
Immunofluorescence Microscopy
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.
Yeast Vector Construction and Expression of Recombinant Maspin
Recombinant maspin was produced in yeast (FLAG expression
system; Sigma) with modifications to the vector. Six histidine residues
were incorporated into the YEpFLAG-1 vector downstream of the FLAG
peptide sequence by overlapping PCR (Fig. 1A)
. The maspin open reading frame (ORF) was amplified from the pGEM
vector by PCR using DNA polymerase (Pfu Turbo polymerase;
Stratagene, La Jolla, CA). To subclone into the YEpFLAG/His-1 vector,
EcoRI and BglII sites were incorporated into both
ends using primers 5'-CGGAATTCATGGATGCCCTGCAACTA-3' and
5'-GCAGATCTTTAAGGAGAACAGAATTT-3'. The maspin ORF was subcloned into the
YEpFLAG/His-1 vector in which the FLAG/His was tagged to N-terminus of
maspin ORF (Fig. 1A)
. The YEpFLAG/His-Maspin construct was transformed
into the Saccharomyces cerevisiae protease-deficient yeast
strain BJ3505 (Sigma) using a lithium acetate transformation method, as
described in the manufacturer’s expression system manual. Yeast
recombinant FLAG/His maspin was expressed in YPHSM medium (1% glucose,
3% glycerol, 1% yeast extract, 8% peptone, and 20 mM
CaCl2). The cultures were grown on a rotary
platform at 175 rpm at 30°C for 72 hours.
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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–ECM Protein Adhesion Assay
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 1 µM 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 x 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.
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Results |
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465-bp RSL
RT-PCR product was amplified from total RNA extracted from all corneal
layers. Control analyses without reverse transcriptase confirmed the
absence of contaminating DNA.
Total RNA from the epithelial and stromal layers was further analyzed
by synthesizing cDNA of the entire 1.2-kb maspin ORF (Fig. 2E)
.
Digestion of the 1.2-kb PCR products with restriction enzymes
BamHI, DdeI, HinDIII, AluI,
and NcoI yielded digestion patterns predicted from the
sequences of human maspin gene (not shown). DNA sequencing confirmed
the identity of the gene. Compared with the human mammary gland maspin
sequence (GenBank, U04313), nucleotide substitutions were found at
positions 196 (A 
G) and 243 (T C). The first substitution
caused a missense mutation that encodes Val instead of Ile at amino
acid residue 66. This Val substitution was previously reported in the
maspin sequences for mouse mammary gland15
(GenBank,
U54705), rat prostate5
(GenBank: U58857), and human
prostate.5
The second substitution was a conservative
change that encodes the same amino acid, a serine at residue 81.
Expression of Maspin in Corneal Stromal Cells in Culture
To further confirm the unexpected finding that corneal stromal
cells express maspin, stromal cell cultures were established
and analyzed by RT-PCR and Western blot analysis for maspin message and
protein, respectively. Total RNA and protein extracts were prepared
from cells of various passages cultured from the same tissue donor. As
shown in Figure 3A
, the 465-bp maspin-specific RT-PCR products were amplified from the
total RNA extracts from both primary and first-passage cells. However,
maspin expression declined to undetectable levels after further culture
propagation (Fig. 3A , passages 2 to 3). Similar to the RT-PCR result,
Western blot analysis showed maspin protein was present in both primary
and first-passage cultures, but was absent in late-passage cells (Fig. 3B)
. As shown in Figure 3C
, the two higher molecular weight bands were
clearly nonspecific, resulting from the secondary HRP-conjugated goat
anti-rabbit IgG antibodies.
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As shown in Figure 5 , pretreatment with exogenous maspin increased attachment of stromal cells to fibronectin, similar to the effect of maspin on carcinoma cells.9 In contrast to maspin-treated carcinoma cells, maspin also increased stromal cell adhesion to type I collagen, type IV collagen, and laminin. Cell attachment to type I collagen was increased by approximately 70%, to laminin by 50%, and to type IV collagen or fibronectin by 30%. Maspin did not increase cell attachment to the nonbiologic substrate BSA, and the homologous SERPIN ovalbumin had no significant effect on cell adhesion.
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Discussion |
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0.1
ml) in the micromolar range (2.5 µM); a level sufficient to be
physiologically significant, because the concentration of maspin
required for biologic activity is in the range of 0.3 to 1
µM.9
10
19 Although maspin is synthesized by corneal keratocytes in situ and by primary and first-passage corneal stromal cells in culture, its expression declines to undetectable levels in later passage cells. Other biosynthetic products also exhibit changes in expression when human corneal stromal cells are propagated in vitro. For example, the synthesis of keratan sulfate proteoglycans dramatically decreases, and the production of the chondroitin-dermatan sulfate proteoglycan decorin and of basement membrane components laminin and perlecan increases in cultured cells.20 Later passage cultured stromal cells have protein expression patterns that are similar to those of cells in wounded corneal stroma. Gene expression of molecules such as matrix metalloproteinases is induced both in cultured stromal cells and wounded corneal stroma.21 22 These characteristics suggest that cultured corneal stromal cells resemble the activated fibroblasts in wounded cornea, and cultured cells are often used as a model of the wound fibroblasts.23 24 The similarity between stromal cells in culture and fibroblasts in corneal wound raises the possibility that wound fibroblasts may, as do late-stage cultured cells, lose maspin expression.
Downregulation of maspin expression in corneal stromal cells after
culturing is reminiscent of the downregulation that occurs in mammary
gland epithelial cells after conversion into carcinoma
cells.4
Both carcinoma cells9
and
late-passage corneal stromal cells (shown herein) respond to exogenous
maspin by increased cell adhesion to ECM. In carcinoma cells, increased
cell adhesion to fibronectin, which appears to be mediated by
upregulation of the fibronectin receptor 5 integrin,9
results in decreased tumor cell migration. Maspin may similarly
regulate corneal stromal cell migration by increasing cell adhesion to
fibronectin.
In contrast to mammary gland tumor cells, maspin also increased corneal stromal cell adhesion to type I collagen, type IV collagen, and laminin. These ECM proteins, together with fibronectin,25 are present in both the normal26 and the wounded corneal stroma,27 28 raising the possibility that maspin plays a regulatory role in stromal wound healing. A possible sequence of events in the wounded stroma is that the conversion of cells to wound fibroblasts results in downregulation of maspin expression, lower maspin levels in the cornea, and therefore a permissive environment for fibroblast migration. As corneal wound healing proceeds and maspin levels in the stroma are restored (perhaps maspin originates from the adjacent healing epithelium), wound fibroblasts may again become stationary due to maspin-induced increases in their adhesion to ECM molecules. Whether maspin regulates stromal wound healing as suggested remains to be determined.
Maspin may also have other functions in the corneal stroma, such as preventing tumor invasion and maintaining avascularity, activities that are consistent with its documented tumor suppressor4 and antiangiogenic properties.10 Although the functions of maspin in the cornea are currently speculative, the data presented herein indicate that the protein is found throughout the cornea, including in the stroma where cells theoretically both produce and respond to maspin.
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Acknowledgements |
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Footnotes |
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Submitted for publication April 25, 2001; revised August 3, 2001; accepted September 14, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: Sally S. Twining, Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. stwining{at}mcw.edu
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2-Macroglobulin levels in normal human and keratoconus corneas Invest Ophthalmol Vis Sci 35,4008-4014This article has been cited by other articles:
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