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Expression of the cysteine proteinase inhibitor cystatin C mRNA in rat eye.

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THE ANATOMICAL RECORD 239:343-348 (1994)
Expression of the Cysteine Proteinase Inhibitor Cystatin C mRNA
in Rat Eye
Department of Cell Biology and Anatomy, Mount Sinai School of Medicine of The City
University of New York, New York, New York
Background: Cystatin C, a naturally occurring inhibitor of
cysteine proteinases, belongs to family 2 of the cystatin superfamily. While
cystatins in general, and cystatin C specifically, are expressed in various
cell types and found in biological fluids, cystatins in ocular structures have
not been investigated. In the present study, the expression of cystatin C
mRNA in the eye of the rat was studied.
Methods: Total RNA was extracted from eyes as well as from pooled
corneae, retinas, lenses, sclerae, and corneae of adult rats. Cystatin C
mRNA was detected in the RNA samples by reverse transcriptase--polymerase chain reaction and Northern blot hybridization. In addition, in situ
hybridizations of formalin-fixedcryostat sections were carried out using a
digoxigenin-labeled cystatin C probe.
Results: Cystatin C mRNA was demonstrated in total RNAs extracted
from the eye, sclera, and retina, but not in RNAs isolated from the cornea
and lens. In situ hybridizations revealed cystatin C mRNA in most of the
stromal cells of the sclera. In the retina, a strong signal was localized in the
outer nuclear layer. The distribution of the reaction product suggested that
in the retina Miiller cells and rod cells are the primary sites of expression of
cystatin C. In addition, some glial cells in the inner nuclear and ganglion
cell layers were stained. No specific signal for cystatin C mRNA was detected in the cornea, lens, iris, ciliary body, and choroid.
Conclusions: In the eye of the rat, significant levels of cystatin C mRNA
are detected in the sclera and retina. In the sclera cystatin C may play a role
in modulating the activities of cysteine proteinases, mostly cathepsins, involved in the turnover and remodeling of the stroma. In the retina, cystatins synthesized and presumably released by Miiller cells and rod cells
may have a protective function against the harmful effects of cysteine proteinases released under physiologic and pathologic conditions.
0 1994 Wiley-Liss, Inc.
Key words: Retina, Sclera, Miiller cell, Cysteine proteinase inhibitor, Cystatin, Polymerase chain reaction, In situ hybridization
Cystatins are naturally occurring inhibitors of cysteine proteinases which include a number of cathepsins, calpains, and multicatalytic and metal-dependent
proteinases (Bond and Butler, 1987). Wide-spread in
plant and animal kingdoms, all known cystatins belong to a n evolutionary superfamily, cystatin, consisting of three families: 1. stefins, 2. cystatins, and 3.
kininogens (Barrett, 1986; Muller-Ester1 and Fritz,
1986; Turk and Bode, 1991).In both the human and the
rat, cystatins (family 2) have been demonstrated in
several cell types a s well a s in biological fluids such as
semen, cerebrospinal fluid, tear, synovial fluid, saliva
(Abrahamson et al., 1986). To our knowledge, however,
the expression of cystatin C , or any other cystatin, in
the eye has not been described. We report here that in
the eye of the rat cystatin C mRNA is expressed; the
primary sites of expression are the retina and sclera.
The functions of cystatin C in the eye remain to be
investigated. Since cysteine proteinases are implicated
in the intra- and extracellular degradation and turnover of proteins, and perhaps in the processing of prohormones and proenzymes, it is plausible to assume
that the inhibitor of these enzymes modulates these
processes under physiologic and pathologic conditions
of the eye.
Received December 3, 1993; accepted March 1, 1994.
Address reprint requests to Tibor Barka, M.D., Mount Sinai School
of Medicine, Box 1007, New York, NY 10029.
Adult female Sprague-Dawley rats were used. The
rats were killed by C 0 2 asphyxiation, and the eyes
were removed for the extraction of RNA and for in situ
hybridization. RNA was also extracted from pooled
lenses, corneae, sclerae, and retinae separated under a
dissecting microscope. The sclerae were scraped from
the choroid but the episcleral tissue was not removed.
Reverse Transcriptase-Polymerase
Chain Reaction (RT-PCR)
For the analysis of the expression of cystatin C gene
a t the mRNA level, we have used reverse transcription
of mRNAs and PCR amplification of cDNA, as well as
Northern blot hybridizations. For both reverse transcription of RNA to cDNA and the subsequent amplification the thermostable rTth DNA polymerase was
used according to the instructions of the manufacturer
(GeneAmpO Thermostable rTth Reverse Transcriptase
RNA PCR Kit, Part No. N808-0069, Perkin Elmer Cetus, Norwalk, CT).
For the PCR amplification the primers were: sense
primer: CYSC-A, 5'-GTAAGCAGCTTGTGGCTGGAA-3' (nt positions 181-201 of rat cystatin C cDNA);
antisense primer: 5'-GCCTTCTTTACTGTCTCCTGGT-3' (nt positions 500-520 of rat cystatin C cDNA)
X16957) (Cole et al., 1989). Using these primers, a
339-bp fragment of cystatin C cDNA was amplified.
The reaction volume for PCR was 100 p.1, and the
concentrations of primers were 0.5 pM each, and those
of the deoxynucleotides 200 pM. The reverse transcriptase reaction mixture contained 250 ng RNA, and
the reaction was for 15 min at 70°C. After a 2 rnin
denaturation at 95"C, each of the 35 or 40 PCR cycles
consisted of denaturation at 95"C, annealing at 55"C,
and extension at 72"C, each for one min. This was followed by a 7 min elongation at 60°C. DNA was electrophoresed in 2% agarose gel (Perkin-Elmer Corp.,
Catalog No. 61408JB) and visualized by ethidium bromide staining.
cording to the instructions of the manufacturer (The
Genius@ System, Boehringer Mannheim, Indianapolis,
IN) as follows: prehybridization for 2 h r and hybridization overnight, both at 55°C. The prehybridization solution consisted of 5 x SSC (1 x SSC = 0.15 M NaC1,
0.015 M trisodium citrate), 50% formamide, 0.02% sodium dodecyl sulfate (SDS), 0.1% N-lauroylsarcosine,
and 2% blocking reagent. The hybridization solution
was the same as the prehybridization solution containing about 20 ng/ml of the digoxigenin-labeled DNA
probe. After hybridization, the membrane was washed
2 x 5 min in 2 x SSC, 0.1% SDS at room temperature,
and then 2 x 15 min in 0.5 x SSC, 0.1% SDS at 65°C.
The detection of the hybridized probe was with antiDIG-alkaline phosphatase antibody. Following a 90
min blocking in 2% blocking solution, incubation with
the antibody (1:5000 dilution) was for 60 min at room
temperature. Alkaline phosphatase activity was demonstrated by incubating with the substrate 5-bromo-4chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) for 2 to 18 hr a t room temperature in the
Preparation of the Digoxigenin-labeled Cystatin C Probe
The digoxigenin-labeled probe was generated in
polymerase chain reactions (PCR) using AmpliTaqB
DNA polymerase (GeneAmpOPCR Core Reagents, Perkin Elmer Cetus, Norwalk, CT) a s described previously
(Barka and van der Noen, 1993).
In Situ Hybridization
The in situ hybridization was performed exactly as
described previously (Barka and van der Noen, 1993).
Briefly, paraformaldehyde-fixed, 8 p thick cryostat
sections were prehybridized and then hybridized to
digoxigenin-labeled cystatin C probe. The hybrids were
detected using alkaline phosphatase-labeled antidigoxigenin antibodies. Alkaline phosphatase activity
was demonstrated with the BCIP-NBT technique.
Expecting a low abundance of cystatin C mRNA in
the eye, we first used a sensitive RT-PCR to detect the
message in the total RNA extracted from the eyes of
Northern Blot Hybridization
rats. Using cystatin C-specific primers, a 339 bp DNA
RNA was isolated by the guanidine isothiocyanate- fragment, as predicted from the sequence of rat cystacesium chloride gradient method (Chirgwin e t al., tin C cDNA (Cole et al., 1989), was amplified, indicat1979) from pooled eyes and from pooled dissected cor- ing the expression of cystatin C mRNA in the eye. As a
neae, lenses, retinas, and sclerae. RNA was also ex- control, we have used RNA from the seminal vesicle
tracted from the seminal vesicle of a n adult male rat. which is known to contain one of the highest concenThe RNA was precipitated twice with ethanol, and was trations of cystatin C in the r a t (Tavera et al., 1990)
dissolved in RNase-free 1mM EDTA, 10 mM NaC1, 10 (Fig. 1A).
In order to analyze the sites of expression of cystatin
mM Tris-HC1, pH 8.0. The RNA was used for both
Northern blot hybridizations and RT-PCR reactions. C mRNA in the eye, RT-PCR was carried out also using
The integrity of the RNA used for the RT-PCR was RNAs isolated from pooled sclerae, corneae, lenses, and
retinas. RT-PCR revealed expression of cystatin C
monitored by agarose gel electrophoresis.
Total RNA (10 p.g) was electrophoresed through 1% mRNA only in the sclera and retina (Fig. 1B). When
agarose, 6% formaldehyde gel, blotted onto positively RNA was omitted from the RT-PCR reaction mixture,
charged nylon membranes (Boehringer Mannheim, In- no amplification was seen.
On Northern blots of RNAs from the seminal vesicle
dianapolis, IN), and crosslinked for 3 min with UV
Stratalinkerm, 1800 (Stratagene, LaJolla, CA). Prior and eye, the digoxigenin-labeled cystatin C probe hyto blotting, the ethidium bromide-stained RNA was vi- bridized to a single mRNA species of about 700 bp (Fig.
sualized and photographed. For Northern blot hybrid- 2).
After establishing by RT-PCR that cystatin C mRNA
izations digoxigenin(DIG1-labeled DNA probes were
used. Hybridization and detection were carried out ac- is expressed in rat eye, and particularly in the sclera
1 2 3 4
Fig. 1. RT-PCR analysis of the expression of cystatin C mRNA. RNA
was extracted from the tissues and subjected to RT-PCR as detailed in
Materials and Methods. Panel A lane 1: eye; lane 2 seminal vesicle;
lane 3: DNA ladder. Panel B: lane 1: lens; lane 2 cornea; lane 3
sclera; lane 4: retina; lane 5 DNA ladder. The arrow indicates the
amplified 339 bp cystatin C fragment. DNA ladder (bp): 1000, 700,
500, 400, 300, 200, 100.
Fig. 2. Expression of cystatin c mRNA in the seminal vesicle and
eye. Northern blot analysis was performed with 10 Fg of total RNA
from the eye and seminal vesicle. RNA was fractionated by electrophoresis through formaldehyde-containing agarose gel (l%), transferred to positively-charged nylon membrane, and probed with digoxigenin-labeled 339 bp DNA probe as described in Materials and
Methods. The arrows indicate the positions (from the top) of origin,
28s rRNA and 18s rRNA. Lane 1: seminal vesicle; lane 2: retina.
and retina, we have further investigated the site(s) of
expression using in situ hybridizations of formalinfixed cryostat sections with-a digoxigenin-labeled cys-
tatin C probe. In accord with the RT-PCR data, in situ
hybridizations revealed that the primary sites of expression of cystatin C mRNA are indeed the sclera and
retina. Unless the incubation for the detection of alkaline phosphatase activity was prolonged, e.g. 18-20 hr,
no signal was detected in any other ocular structure.
Figure 3 is a photomicrograph of a formalin-fixed
cryostat section adjacent to those used for the in situ
hybridizations and stained with hematoxylin-eosin. It
indicates the degree of structural preservation and
serves for orientation.
In the sclera, the hybridization signal was seen in
the cells of the stroma (Figs. 4-6). Most, but not all
cells gave the reaction. In general, cells in the episclera
were negative. In the sclera-cornea transition (limbus)
a few cells were stained. However, the distribution and
frequency of cells giving the signal in different parts of
the sclera were not studied.
In the retina, reaction products (formazan deposits)
were seen mostly in the outer nuclear layer (Figs. 4,5,
7 and 8). There were, however, cells frequently in
groups, displaying the signal in the inner nuclear layer
(Figs. 5 and 8), and occasional cells in the ganglion
layer. In the outer nuclear layer, the staining seen after 2-3 h r incubation outlined slender cytoplasmic processes not uncommonly surrounding a nuclear halo
reminiscent of the shape and distribution of the cytoplasm of the Miiller cells (Figs. 7 and 8). After prolonged incubations for alkaline phosphatase activity,
or with high probe concentrations, the signal became
very intense over the entire outer nuclear layer (Fig.
5). Under such conditions, the structural details were
diEcult to discern but formazan deposits appeared to
surround and outline the densely packed nuclei of the
inner nuclear layer. This pattern of staining is compatible with a localization of the signal to rod cells.
In the retina, no specific signal was discerned in the
pigment epithelium (Fig. 4 and 5 ) , in the outer segments of rod and cone cells (Figs. 4,5and 7), and in the
bipolar, horizontal, and ganglion cells (Fig. 7).
No signal was observed in any other ocular structures such as the cornea (Fig. 9))choroid (Figs. 4,5,and
61,iris, ciliary body, or the lens (data not shown). Although after prolonged incubation for the demonstration of alkaline phosphatase activity some formazan
deposits were seen in these structures, the specificity of
such a reaction is questionable.
When the sections were treated with RNAse prior to
hybridization, or the probe was omitted from the hybridization mixture, no specific staining occurred. Such
control preparations were similar in appearance to the
negative areas in the preparations shown (e.g. cornea
in Fig. 9).
Using RT-PCR and Northern, and in situ hybridizations we have established that cystatin C mRNA is
expressed in rat eye, and that the primary sites of expression are the sclera and the retina.
The sclera was one of the least expected sites of expression of cystatin C mRNA. Among the cellular systems of the eye, the stromal cells of the sclera have
been most neglected by morphologists and physiologists alike. As a matter of fact, in a detailed electron
Fig. 3. Photomicrograph of a cryostat section stained with hematoxylin-eosin. It illustrates the structural preservation obtained and
serves for orientation. SC = sclera; CH = choroid; 0s = outer segment; ON = outer nuclear layer; IN = inner nuclear layer; IP =
inner plexiform layer; GC = ganglion cell layer.
is a similar to that shown on Fig. 4, except hybridization was performed with another batch of probe and incubation in full-strength
medium was for 20 hr. The reaction is very intense in cells in the
outer nuclear layer, but stained cells are also seen in the inner nuclear layer.
Figs. 4-9. Photomicrographs of sections after in situ hybridization
for cystatin C mRNA. Formalin-fixed cryostat sections were hybridized with a 339-bp digoxigenin-labeled DNA probe specific for cystatin C as described in Materials and Methods.
Fig. 6. Reaction is seen in scleral cells. Cells in the choroid are
devoid of reaction product. SC = sclera; CH = choroid.
Fig. 4. Localization of the reaction product in sclera cells and in cells
in the outer nuclear layer of the retina. All other structures are negative.
Figs. 7 and 8. Portions of the retina in preparations after relatively
short incubation. Cells in the outer nuclear layer are stained. The
reaction product outlines cellular processes (long arrow on Fig. 8 ) or
surrounds nuclei (e.g. arrows on Fig. 7 and curved arrow on Fig. 8).
Single cells or group of cells are also seen in the inner nuclear layer
(short arrows). These cells are considered to be glia cells.
Fig. 5. Localization of the reaction product in sclera cells and in cells
in the outer and inner nuclear layers of the retina. This preparation
Fig. 9. Cornea. No signal is seen in the epithelium (El or stroma.
Scale bar: 20 Fm.
microscopic study of the sclera by Schwarz (1953)and
in text-books (e.g. Simpson and Savion, 1982) scleral
cells are not mentioned. Scleral cells are generally considered to be fibroblasts, perhaps with the exception of
the scleral spur cells which are contractile and have
the characteristics of myofibroblasts (Tamm et al.,
1992, 1993). In situ hybridizations revealed cystatin C
mRNA in the majority but not all of the scleral cells,
suggesting that they are heterogeneous. Knese (1971)
already described, on the basis of morphologic criteria,
two types of cells in rat sclera.
In the last years interest in the sclera has been stimulated by experimental data on myopia induced by visual deprivation. There is accumulating evidence that
scleral changes, including scleral growth, may underlie
myopia induced by visual deprivation (Funata and
Tokoro, 1990; Christensen and Wallman, 1991). These
scleral changes and growth are under retinal influences, although the mechanisms involved are not
known (Wallman et al., 1987; Wallman, 1993). In the
context of the data on the role of sclera in the development of axial myopia, the presence of cystatin in scleral
cells is of considerable interest. Growth and remodeling of the sclera certainly involve not only synthetic
but also catabolic processes resulting in degradation of
collagen fibers and components of the matrix by enzymes released by scleral cells, and, under pathologic
conditions, inflammatory cells. Several lysosomal
cathepsins such as cathepsins B, H, L, and N are cysteine proteinases, and are known to degrade, in concern with other proteolytic enzymes, collagen (e.g. Maciewicz et al., 1987, 1990) and other proteins. Since
cystatins control the activity of these enzymes, they are
likely to play a role in the turnover and remodeling of
the scleral stroma. While scleral cells express cystatin
C mRNA, stromal cells in the cornea, also considered to
be fibroblasts, do not. This observation is a further indication of specialization of some of the scleral cells.
Both RT-PCR and in situ hybridizations revealed
cystatin C mRNA in the retina, where most of the signal was seen in the outer nuclear layer. Because of the
complex morphology of this layer, it is difficult to establish the exact cellular location of the hybridization
signal. Examinations of preparations displaying a
weak signal after relatively short incubations, suggest
that Muller cells express cystatin C mRNA. This conclusion is based on the correspondence of the localization of formazan deposits and the shape and structure
of Muller cells (Wolter, 1961) such as a n irregular
shape of cell body, often showing a concave outline
around nuclei in the outer nuclear layer, and long
processes radiating from the cell body and occupying
the thickness of the retina. One ought to consider,
however, that mRNA molecules bound to ribosomes
may not be found in the long processes (fibers) (e.g. in
the inner nuclear layer), and that the distribution of
mRNAs is compromised by the fixation and freezing
process. We have observed, in preliminary experiments, that cultured Muller cells contain cystatin C
and express cystatin C mRNA, supporting the notion
that Muller cells are a source of cystatin C in the retina
(Barka and van der Noen, unpublished observations).
Cells or group of cells occasionally seen in the inner
nuclear layer, and a few single cells in the ganglion
layer, which were also stained, are most likely glial
cells. The expression of the cystatin C gene by glial
cells in the retina would be in line with the findings of
Zucker-Franklin et al. (1987) who showed that microgliaUastroglia1 cells isolated from mouse brain constitutively secrete cystatin C. Although the data indicate
that supporting cells express cystatin C in the retina,
after prolonged incubations the signal became very
strong over the entire outer nuclear layer. Such a
strong signal cannot be ascribed to supporting cells
alone, and it implies that the cystatin C gene is expressed also by rod cells.
Since cystatin C is secreted from the cells, it is reasonable to assume that the inhibitor released plays a
protective role against cysteine proteinases which may
be also released under physiologic, but particularly under pathologic (inflammation) conditions. In addition,
cystatins may also modulate the phagocytic function of
Miiller cells (Nishizono et al., 1993). Intraocular injection of E64C, a potent inhibitor of cysteine proteinases,
led to the accumulation of lipofuscin-like substances in
lysosomes in the retina (Ivy et al., 1990), supporting
the notion that cystatins may modulate the functions of
phagolysosomes. These problems are amenable to experimental analyses using cultured Muller cells.
It has been reported that the cystatin proteinase inhibitor E64 prevented or ameliorated experimental
cataract formation (Shearer et al., 1991; Azuma et al.,
1992; Kadoya et al., 1993). We have found no cystatin
C mRNA in the lens. While E64 and its derivatives are
strong inhibitors of all cysteine proteinases, including
calpains, cystatins do not or only weakly inhibit
calpains. This suggests that if cysteine proteinases and
their inhibitors play any role in some form of cataract
formation (e.g. induced by calcium ionophore), the system involved is the calpains (Kadoya et al., 1993) and
calpastatin, and not cystatin C. However, the expression of other types of cystatins, e.g. stefins, in the lens
and in the eye in general, remains to be investigated.
Cystatin C is expressed in most, if not all, human
tissues examined (Abrahamson et al., 1987). The level
of expression varies, however, and in several tissues
cystatin C mRNA is barely detectable by Northern blot
hybridization. The expression of cystatin C mRNA in
the rat has not been studied systematically, but the
concentration of cystatin C was measured in different
tissues by radioimmunoassay (Tavera et al., 1990). The
highest levels of the protein were found in the seminal
vesicle, cerebrum, pituitary gland, cerebellum, and
kidney cortex. Although cystatins, to our knowledge,
have not been demonstrated in ocular tissues, cystatin
C has been localized immunocytochemically in neurons
in human brain (Bernstein et al., 1988; Lofberg et al.,
1981).Cystatin C is expressed also in rat brain (Cole et
al., 19891, and cystatins were isolated from the brain of
the rat (Kopitar et al., 1983; Marks et al., 1988). In
addition, the cerebrospinal fluid contains cystatin C in
a concentration exceeding that of the plasma (Lofberg
and Grubb, 1979; Grubb and Lofberg, 1985). The source
of cystatin C in the cerebrospinal fluid is most likely
the choroid plexus. Tears also contain cystatins (Abrahamson et al., 1986; Barka et al., 1991), and cystatins
have been localized immunocytochemically in human
lacrimal gland and in the exorbital lacrimal gland of
the rat (Takahashi et al., 1992).
A mutation of the cystatin C gene, resulting in a
single amino acid substitution, glutamine for leucine
at position 68 in the cystatin C polypeptide chain, is
responsible for the hereditary cystatin C amyloid angiopathy (hereditary cerebral hemorrhage with amyloidosis) (Grubb et al., 1984; Ghiso et al., 1986; Jensson
et al., 1987; Abrahamson et al., 1992). This dominantly
inherited disease is characterized by deposition of amyloid in most investigated tissues. Whether the eyes are
involved in this fatal disease has not been investigated.
A hereditary disease involving alteration of cystatin
C, and the roles cystatins play in physiologic and
pathologic processes, including inflammation, autoimmune diseases, and metastasis of tumors, underscore
the importance of studying cystatins, and other proteinase inhibitors, in ocular tissues of humans and
other species.
This work was supported by Grant HL39419 from
the National Heart, Lung, and Blood Institute.
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expressions, inhibitors, proteinase, cystatin, rat, cysteine, eye, mrna
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