THE ANATOMICAL RECORD 239:343-348 (1994) Expression of the Cysteine Proteinase Inhibitor Cystatin C mRNA in Rat Eye TIBOR BARKA AND HENDRIKA VAN DER NOEN Department of Cell Biology and Anatomy, Mount Sinai School of Medicine of The City University of New York, New York, New York ABSTRACT 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. 0 1994 WILEY-LISS. INC. 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. 344 T. BARKA AND H. MATERIALS AND METHODS Animals 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) (GenBank, LOCUS: RATCYSC, ACCESSION: 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. VAN DER NOEN 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 dark. 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. RESULTS 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 345 CYSTATIN IN THE EYE A 1 2 3 1 2 3 4 5 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. 1 2 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). DISCUSSION 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. CYSTATIN IN THE EYE 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 347 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 348 T. 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