Journal if Neurochemi~try Raven Press, Ltd., New York 0 199 I International Society for Neurochemistry
Changes Induced in Astrocyte Cathepsin D by Cytokines and Leupeptin *tSJ. N. Whitaker, TP. K. Herman, tS. M. Sparacio, TS. R. Zhou, and t$E. N. Benveniste *Neurology and Research Services of the Birmingham VA Medical Center, and the Departments of ?Neurology and $Cell Biology of ihe University of Alabama at Birmingham, Birmingham, Alabama, U.S.A.
Abstract: Cathepsin D is widely, but unevenly, distributed among cells and is capable of degrading a number of neural peptides and proteins. The present study was undertaken to examine the level of cathepsin D in astrocytes that might be relevant to its induction in inflammatory demyelination. Primary astrocytes were cultured from neonatal rat cerebrums according to the method of McCarthy and de Vellis. Based on staining for cell markers, cultures were >95% astrocytes and 95% of cells present in the culture. The cathepsin D immunoreactivity varied in intensity among cells and was distributed in a granular, presumably lysosoma1 pattern. J. N e ~ i r d w r n . .VoL S 7 , No. 2, 1991
We examined whether cathepsin D levels in astrocytes could be modulated with a variety of stimuli, including cytokines. An inherent problem in the study of primary astrocytes, as compared with an astroglial cell line, is their variation among different batches. This variation was noted in astrocyte cathepsin D even after relating the immurioreactive cathepsin D level to protein. The values of 0.3-0.9 ng of cathepsin D/pg of protein are similar to results obtained on rat neural tissue specimens (Snyder and Whitaker, 1983; Whitaker and Crowley, 1983). Because of this variation, for comparisons and statistical analysis, it was necessary to standardize internally the measurements of cathepsin D changes. Thus, the measurement of cathepsin D, in nanograms per micrograms of protein, in cells grown in culture medium alone, was assigned a value of 1.O, and induced changes were expressed as a ratio (see legend of Fig. 2 for additional details). Exposure of cultured neonatal rat astrocytes to leupeptin led to a rise in cellular cathepsin D immunoreactivity (Fig. 2). Little change occurred at 16 and 24 h, but an increase of I .4to 2.0-fold from the control level of approximately 0.5 ng of cathepsin D/pg of protein could be detected by 48-72 h of culture in a dose-dependent fashion over a concentration range of leupeptin ( 1- 100 pg/ml). A concentration of 2 5 pg/ml of leupeptin was shown to be optimal for stimulating the increase in cellular levels of cathepsin D over 48 h of culture. An increase in cathepsin D immunoreactivity was clearly evident when astrocytes were cultured under serum-free conditions. Results of several studies supported the conclusion that the specific activity of astrocyte cathepsin D rose after leupeptin treatment. Total protein content did not decrease and actually increased in leupeptintreated compared with control cultures (Fig. 2A); hence, the increase in cathepsin D was due to increased cathepsin D synthesis and not a decline in cell protein. As determined by the two-way analysis of variance, the difference in the increase in cathepsin D between 16 and 48 h in the presence of leupeptin was significant ( p < 0.01). The leupeptin-inducedincrease in cathepsin D between 16 and 24 h as well as between 24 and 48 h was also significant ( p < 0.05). Repeat experiments revealed a mean (fSD) increase in the ratio, as illustrated in Fig. 2C, at 48 h of 1.7 f 0.1 (n = 3) and at 72 h of 2.1 k 0.4 (n = 7). Supernatants from cultured astrocytes revealed no change in cathepsin D immunoreactivity after treatment with leupeptin, implying no change in secretion of cathepsin D effected by leupeptin. The observed increase in cathepsin D by leupeptin was not an artifact due to any effect of leupeptin on the radioimmunoassay, in that leupeptin added directly to the immunoassay tubes at concentrations of 25- 100 pg had no effect on the assay results. Cathepsin D immunoreactivity detected in control and leupeptinexposed astrocytes demonstrated immunochemical parallelism (Whitaker et al., 198l), indicating that the cathepsin D produced in basal and leupeptin-stimulated cultures was identical immunochemically.
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FIG. 1. Cathepsin D immunofluorescenceof astrocytes. Astrocytes were plated on coverslips, cultured for 2 days, washed, and fixed for 10 s in cold acetone. The cells were stained with a polyclonalrabbit antibody (R164) to rat cathepsin D (1 :loo),then with fluoresceinated goat anti-rabbit immunoglobulin (1:20).The cells were visualized by fluorescence microscopy for total number of cells (nuclei observed with the Hoechst stain) (top) and cells immunoreactive for cathepsin D (bottom); X800.
With leupeptin as a positive control, a number of cytokines known to modulate gene expression in astrocytes (Benveniste, 1988) and other substances such as LPS, calcium ionophore, and substance P were tested for their ability to influence cathepsin D levels. At all concentrations tested and for up to 72 h of incubation, neither human recombinant TNF-a nor rat recombinant IFN-y increased cathepsin D levels compared with untreated control cells. No effect was noted when IFNy and TNF-a were added simultaneously at concentrations of 80 U/ml and 50 ng/ml, respectively, to astrocyte cultures. LPS ( 10 pg/ml), substance P ( 1- 1000 nM), IL-lp (0.1-10 ng/ml), and calcium ionophore (5 p M ) effected no change in astrocyte cathepsin D content at 48 h. As previously noted (Whitaker, 1980), the enzyme assay of cathepsin D is much less sensitive than the immunoassay. Cathepsin D enzyme activity, however, could be detected in the cultured astrocytes. Control astrocytes had enzyme activity that solubilized 5.4-
5.6%, approximately O.O4%/pgof protein, of the added substrate after culture for 48-72 h. This increased 1.2and 1.6-fold after exposure for 48 and 72 h, respectively, to leupeptin 2 5 pg/ml. The cathepsin D enzyme activity and its increase on leupeptin exposure were inhibited by pepstatin (8 pglml), indicating that the enzyme activity was in fact due to cathepsin D. Because of the increased cathepsin D appearing after exposure of rat astirocytes to leupeptin, quantitative changes in cathepsin D mRNA were studied. Northern blot analysis demonstrated that cathepsin D mRNA transcripts of the appropriate size (2.2 kb) were present in cultured astrocytes, and specifically increased by leupeptin (Fig. 3). Kinetic analysis of cathepsin D mRNA expression was examined by slot blot analysis. The mean k SD of two separate experiments is represented graphically in Fig. 4A. Cathepsin D mRNA began to increase between 12 and 16 h, reached a maximal induction of a fourfold increase over control at 24 h, and declined at 48 h. Cyclophilin mRNA levels J. Neurochem.. Val. 57, No. 2, 1991
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FIG. 3. Cathepsin D rnRNA expression in astrocytes. Northern blot analysis of cathepsin D rnRNA. Astrocytes were incubated with or without leupeptin(25 pg/ml) for 16 h. Samples (15.0 pg) of total cellular RNA isolated from unstimulatedrat astrocytes (lane A) and leupeptin-stimulatedrat astrocytes (lane B) were denatured with formaldehyde for 15 rnin at 55°C. The RNA was size-fractionatedby electrophoresis through a 1.O% agarose gel containing ethidium bromide for visualization of 28s and 18s ribosomal RNA. The RNA was then transferred to nitrocellulose in 2OX SSC. The blot was probed with cathepsin D cDNA and exposed at -70°C for 72 h to Kodak X-Omat AR film plus two intensifying screens. Note the pronounced increase in the 2.2-kb RNA species after exposure to leupeptin.
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Leupeptin (pglml) FIG. 2. Changes in cathepsin D levels in astrocytes exposed to leupeptin. A: Cells were cultured in the presence of 0-100 pg/ml of leupeptinfor 48 h at 37"C, and cell homogenates were analyzed for amounts of total protein (0)and immunoreactive cathepsin D (m). 8: The relative amount of cathepsin D (0)increasedover control at all concentrations of leupeptin tested. C Astrocytes cultured with 0-1 00 pg/ml of leupeptin for 16 ( O ) , 24 (A),and 48 (0)h at 37°C revealed increases in specific immunoreactivityof cathepsin D. Cathepsin D was quantified by radioimmunoassay, related to the amount of total protein present, and graphed as a ratio of the amount of cathepsin D (in nanograms per micrograms of protein) in leupeptin-treatedcultures to the amount of cathepsin D in cultures with no leupeptin. For results shown in (C), the control values were 0.67-0.93 ng cathepsin D/pg of protein.
were examined as a control gene as levels of this gene are generally not altered on cell stimulation (Bethea et al., 1990; Melner et al., 1990). The cathepsin D blots were stripped and reprobed for cyclophilin mRNA. There was no increase in cyclophilin mRNA at 12 or J . Nrurochem., Vol. 57, No. 2,I991
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Time (hours) FIG. 4. Kinetic analysis of cathepsin D and cyclophilin mRNA expression. A Slot blot analysis of cathepsin D mRNA. Total cellular RNA from astrocytes cultured without (0)or with leupeptin (25 pg/ml) (0) for 12, 24, and 48 h was diluted over a range of 6.01.5 pg, blotted onto nitrocellulosepaper, cross-linked,and probed with a labeled cathepsin D cDNA probe. The blot was exposed at -70°C for 24 h to Kodak X-Omat AR film plus two intensifying screens. mRNA values are expressed in arbitrary units as determined from densitometricscanning of autoradiographs.The mean f SD of two experiments is represented graphically. 8: Slot blot analysis of cyclophilin mRNA. Slot blots probed for cathepsin D mRNA (A) were stripped and reprobed with labeled cyclophilin cDNA probe. The blot was exposed at -70°C for 14 h to Kodak X-Omat AR film plus two intensifying screens. Cyclophilin mRNA from astrocytes cultured without (0)or with (0) leupeptin was expressed as for cathepsin D mRNA. The mean SD of two experiments is representedgraphically.
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48 h. An increase ( 1.5-fold) in cyclophilin mRNA was noted at 24 h on stimulation with leupeptin (Fig. 4B). Thus, both cyclophilin and cathepsin D mRNA expression are modulated by leupeptin, although cathepsin D levels are enhanced to a greater extent. The molecular features of cathepsin D in astrocytes were examined by immunoblotting of electrophoretically transferred, separated astrocyte proteins (Fig. 5). The major band of immunoreactive cathepsin D in astrocytes had a molecular mass of 40-42 kDa (Fig. 5B), identical in migration with that of cathepsin D isolated from whole rat brain (Whitaker, 1981). The dominant band of immunoreactive cathepsin D in cultured astrocytes was broad. Although this broad band could be comprised of several closely migrating proteins of similar size, it is more likely the result of the low dilution of anti-cathepsin D antiserum selected to demonstrate other bands of lower intensity. With increasing amounts of protein applied to the gels, other bands of approximately 68, 45, and 35 kDa reactive
FIG. 5. SDS-PAGE and immunoblot analysis of cathepsin D immunoreactivity in cultured primary astrocytes. Primary astrocytes (1 X 10’) were cultured in serum-free media alone (lanes 1 and 2) or with leupeptin at a concentration of 5 pg/ml (lanes 3 and 4), 25 pg/ml (lanes 5 and 6), or 125 pg/ml (lanes 7 and 8) for 48 h at 37°C.The medium was removed, the cell pellet placed in 200 pl of sample buffer, and processed for SDS-PAGE in 15% gels. Forty micrograms (lanes 1 , 3,5,and 7)or 20 pg (lanes 2, 4, 6, and 8) of protein was applied in each lane. The gel was stained with Coomassie Blue (A) or processed for electrophoretic transference of separated proteins, which were subsequently reacted with antirat cathepsin D (B). Molecular weights (X10-3) of markers are indicated. The arrow points to a 45-kDa molecular band that has been tentatively identified as procathepsin D.
with anti-cathepsin D were noted (Fig. 5B). None of these bands corresponded with the main bands detected by staining with Coomassie Blue (Fig. 5A). The pattern of cathepsin D immunoreactivity in leupeptin-treated astrocyteswas basically the same as controls except for a band, estimated at 45 kDa molecular mass, migrating just above the broad cathepsin D band that was more abundant in astrocytes treated with 25 and 125 pg/ml of leupeptin (Fig. 5B, lanes 5-8, arrow). The nature of this 45-kDa immunoreactive protein is unknown, but it has tentatively been identified as the procathepsin D. Pulse-labeling experiments will be required to validate this identification.
DISCUSSION The events and control of intracellular proteolysis in astrocytes are poorly understood but presumably involve lysosomal proteinases such as cathepsin D, which astrocytes contain in vivo (Snyder et al., 1985). The present study demonstrates that cathepsin D can be detected in cultured rat astrocytes at a concentration of 0.3-0.9 ng/pg of protein, a level approximately the same as some rat brain nuclei (Whitaker and Crowley, 1983), spinal cord (Snyder and Whitaker, 1983), and sciatic nerve (Whitaker et al., 1983a,b). Although astrocyte cathepsin D could be increased by leupeptin, cytokines such as recombinant rat IFN-y, TNF-a, a combination of IFN--/ and TNF-a, and IL- 1/3, did not increase astrocyte cathepsin D protein, nor did substance P, LPS, calcium ionophore, or LPS plus calcium ionophore. In a previous study, it was shown that cultured rat primary astrocytes, exposed to conditioned media derived from human peripheral blood mononuclear cells stimulated with either concanavalin A or phytohemagglutinin (PHA), have an increase in cathepsin D enzyme activity. PHA alone, purified human IL- 1, purified human IL-2, and recombinant human IFN-y produced no change (Bever et al., 1989). The present investigation also revealed no stimulatory effect of a variety of cytokines on astrocyte cathepsin D, implying that other types, amounts, or combinations of cytokines are responsible for the stimulatory effects observed (Bever et al., 1989).Leupeptin also increased cathepsin D mRNA levels in primary astrocytes. Whether the effect of leupeptin is exerted at the transcriptional or posttranscriptional level is as yet unknown. Thus, cathepsin D is inducible in astrocytes, but the normal or pathological factors capable of its induction in vivo remain uncertain. Leupeptin also appeared to alter cellular processing of cathepsin D in increasing the amount of cellular procathepsin D, similar to that observed in cardiac fibroblasts (Samarel et al., 1989), without increasing the amount secreted into the culture medium. Similar to other lysosomal proteinases, the mRNA of cathepsin D leads to the synthesis of a preprocathepsin D that loses its 20 residue signal peptide and leaves the Golgi apparatus as the glycoprotein procathepsin D (EnckJ. Neurorhem., Vol. 57, No. 2. 1991
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son, 1989). Procathepsin D, believed to be inactive as an enzyme, loses its 44-residue propiece en route to or in the lysosome. Its single chain has a molecular mass of 40-45 kDa and may be cleaved further, in a speciesdependent fashion, to a heavy chain of 30 kDa and a light chain of 15 kDa (Erickson, 1989). Rats have mainly the single chain form of cathepsin D (Whitaker, 1981), but may have a minor portion of the two-chain variety (Yonezawa et al., 1988). The mechanism for the conversion of procathepsin D to cathepsin D is uncertain, but has been postulated to be a result of leupeptin-sensitive proteinases (Hentze et al., 1984; Gieselmann et al., 1985) or of autocatalysis (Conner, 1989). In the synthesis of cathepsin D, the proform may have a molecular mass of 46-52 kDa (Hasilik and Neufeld, 1980; Westley and May, 1987). Higher molecular weight bands of immunoreactive cathepsin D of different sizes have been noted in human promonocytes (Stein et al., 1987), human skin fibroblasts (Hasilik and Neufeld, 1980), and C6 glioma cells (Snyder and Whitaker, 1988). Their identity and relationship to the other molecular species of cathepsin D are unknown. Similar to the primary astrocytes studied in this investigation, cathepsin D is present in C6 glioma cells (Snyder and Whitaker, 1988). Cathepsin D in C6 glioma cells appears in different sizes besides the dominant 42-kDa form and can be modified by cyanate and monensin presumably through an effect on the Golgi apparatus (Snyder and Whitaker, 1988). The cDNA for cathepsin D has been obtained from a human hepatoma library (Faust et al., 1985) and by sequencing an mRNA induced in a human breast cancer cell line, MCF7, by estradiol (Westley and May, 1987). The cathepsin D gene has been localized to the pl5 band of chromosome 1 1 in the MCF7 breast cancer cell line (Augereau et al., 1988). The mRNA of cathepsin D has a size of 2.1-2.2 kb and contains sequences for the amino terminal pre- and pro-portions of the proteinase (Faust et al., 1985). The size of the cathepsin D mRNA in astrocytes reported in the present investigation is the same (2.2 kb). Although cathepsin D mRNA seems to be rather stable in cultured cells (Faust et al., 1985), it may be induced in selected cells by estradiol, epidermal growth factor (Cavailles et al., 1989), and calcitrol (Redecker et al., 1989). Cathepsin D mRNA levels are relatively uniform in different rat tissues (San Segundo et al., 1986), even though cathepsin D localization may vary widely among different cells of rat organs (Whitaker and Rhodes, 1983). Cathepsin D mRNA showed no changes in a myogenic rat cell line cultured in the presence of serum (Colella et al., 1986). Leupeptin is a naturally occurring, water-soluble proteinase inhibitor derived from streptomyces (Umezawa, 1976). Leupeptin is small, having a molecular weight of 475, binds well to trypsin and cysteine proteinases, and is effective as an inhibitor for many proteinases including plasmin, trypsin, papain, calpain (Umezawa, 1976;Vitto and Nixon, 1986),the cysteine
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proteinases including cathepsins B, H, and L (Umezawa, 1976), and the trypsin-like site on multicatalytic proteinase (Rivett, 1989). Leupeptin has an effect, usually presumed to be a result of its properties as a proteolytic inhibitor, on several cell functions. These include a reduction of the PHA-induced proliferation of guinea pig lymphocytes (Saito et al., 1972), inhibition of the growth of normal and polyoma-transformed baby hamster kidney cells (McIlhinney and Hogan, 1974), and severe growth retardation and abnormal development of rat conceptuses in vitro (Freeman and Brown, 1985). Cerebral intraventricular injection of leupeptin can block olfactory discriminitive learning interpreted to be the result of inhibition of proteinases involved in memory formation (Staubli et al., 1985). Leupeptin may have biological properties other than those of a proteolytic inhibitor. Leupeptin added to primary cultures of adult rat hepatocytes predictably diminished cathepsin D activity but unexpectedly led to an induction of an acid proteinase, partially inhibited by pepstatin (Tanaka et al., 1979). Similar to our findings with cultured primary rat astrocytes, cathepsin D activity was maximally stimulated in rat hepatocytes by 25-50 pg/ml of leupeptin. Although the detection was by enzymatic activity rather than by immunoassay, the leupeptin-stimulated rat hepatocyte responded more quickly and to a greater extent than did the cultured astrocytes. The induction of the acid proteinase activity by leupeplin was not a property of all small peptide inhibitors of microbial origin (Tanaka et al., 1981) and could be prevented by inhibition of RNA and protein synthesis (Tanaka et al., 1979). The exact nature of the acid proteinase induced by leupeptin in rat hepatocytes was not delineated; however, it was later shown to have features of a cysteine proteinase similar to, but not identical with, cathepsin L (Tanaka et al., 1981;Tanaka et al., 1984). The present results provide proof at the RNA, protein, and enzyme levels that the proteinase induced by leupeptin in astrocytes is cathepsin D. The cathepsin D mRNA was increased between 12 and 16 h, followed by a cathepsin D immunoreactivity increase at 24-48 h. The fact that leupeptin induced cathepsin D and another proteinase indicates that some of the cellular changes attributed to leupeptin on the basis of its property as a proteinase inhibitor may be due in part to its effect on inducing proteinases and, possibly, other cellular constituents. The role of cathepsin D and other proteinases in the variety of functions attributed to astrocytes remains to be defined. Some naturally occurring proteinase inhibitors, especially a,-antitrypsin, a2-macroglobulin, and antithrombin 111, stimulate astrocyte proliferation and a small increase in1 glutamine synthetase (Perraud et al., 1988). Astrocyles may serve as antigen-presenting cells, a function that requires proteolysis, if first induced by IFN-7 (Fontana et al., 1984), virus (Massa et al., 1986),or I F N y plus TNF-a (Benveniste et al., 19898), to express major hlistocompatibility complex class I1 antigens. This is suggested as the principal way in which
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astrocytes might initiate or support an in situ immune response in inflammatory demyelination (Fontana et aI., 1987). Cathepsin D is a likely participant in antigen processing (Diment and Stahl, 1985; van Noort and van der Drift, 1989). As shown by the present study, one of the conditions for induction of major histocompatibility complex class 11, that is, IFN-./ and TNF-a stimulation, did not lead to an increase in astrocyte cathepsin D. The actions of cathepsin D or the quantitative levels needed in the antigen-processing function of astrocytes are unknown. The induction of astrocyte cathepsin D presumably requires biological factors, alone or in combination, that are not yet identified. Acknowledgment: This research was supported by the Research Program of the Veterans Administration (J.N.W.) and by National Institutes of Health Grant A1 27290 (E.N.B.). We thank Mr. Alan Clayton for technical assistance, Mr. Albert Tousson for assistance in fluorescence microscopy, Mr. Feng Sun for assistance in the statistical analysis, and Ms. Joanne Cage for assistance in preparation of the manuscript.
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a protease inhibitor, inhibits the PHA-stimulated DNA synthesis in guinea pig blood lymphocytes. Jpn. J. Exp. Med. 42, 509511. Samarel A. M., Ferguson A. G., Decker R. S., and Lesch M. (1989) Effects of cysteine protein inhibitors on rabbit cathepsin D maturation. Am. J. Physiol. 157, C1069-C1079. San Segundo B., Chan S. J., and Steiner D. F. (1986) Differences in cathepsin B mRNA levels in rat tissues suggest specialized functions. FEBS Lett. 201, 251-256. Satav J. G. and Katyare S. S. (1981) Thyroid hormones and cathepsin D activity in the rat liver, kidney and brain. Experientia 37, 10101. Snyder D. S. and Whitaker J. N. (1983) Postnatal changes in cathepsin D in rat neural tissue. J. Neurochem. 40, 1 161-1 170. Snyder D. S. and Whitaker J. N. (1988) Alterations of the posttranslational processing of a lysosomal enzyme in C6glioma cells. J. Neurosci. Res. 20, 73-83. Snyder D. S., Simonis S., Uzman B. G., and Whitaker J. N. (1985) Rat neural tissue cathepsin D: ultrastructural immunocytochemistry. J. Neurocytol. 14, 579-596. Stiubli U., Baudry M., and Lynch G. (1985) Olfactory discrimination learning is blocked by leupeptin, a thiol protease inhibitor. Brain Res. 337, 333-336. Stein M.. Braulke T.. von Finura K.. and Hasilik A. (1987) , , Effects of differentiation-inducing agents on synthesis, maturation and secretion of cathepsin D in U937 and HL-60 cells. Biol. Chem. Hoppe Seyler 368,413-418. Tanaka K., Ikegaki N., and Ichihara A. (1979) Induction of hemoglobin-hydrolase activity by the thiol-protease inhibitors leupeptin and antipain in adult rat liver cells in primary culture. Biochem. Biophys. Res. Commun. 91, 102-107. Tanaka K., Ikegaki N., and Ichihara A. (1981) Effects of leupeptin and pepstatin on protein turnover in adult rat hepatocytes in primary culture. Arch. Biochem. Biophys. 208,296-304. Tanaka K., Ikegaki N., and Ichihara A. (1984) Purification and characterization of hemoglobin-hydrolyzing acid thiol protease induced by leupeptin in rat liver. J. Biol. Chem. 259, 5937-5944. Traugott U. (1988) Multiple sclerosis: complex role of astrocytes in lesion development, in The Biochemical Pathology ofAstrocytes (Norenberg M. D., Hertz L., and Schousboe A., eds), pp. 261272. Alan R. Liss, New York.
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Umezawa H. (1976) Structures and activities of protease inhibitors of microbial origin, in Methods in Enzymology, Vol. XL V:Proteolytic Enzymes, Purl B (Lorand L., ed), pp. 678-695. Academic Press, New York. van Noort J. M. and van der Drift A. C. M. (1989) The selectivity of cathepsin D suggests an involvement of the enzyme in the generation of T-cell epitopes. J. Biol. Chem. 264, 14159- 14164. Vitto A. and Nixon R. A.(1986) Calcium-activatedneutral proteinase of human brain: subunit structure and enzymatic properties of multiple molecular forms. J. Neurochem. 47, 1039-105 1. Westley B. R. and May F. E. B. (1987) Oestrogen regulates cathepsin D mRNA levels in oestrogen responsive human breast cancer cells. Nucleic Acid:r Res. 15, 3773-3786. Whitaker J. N. (1980) Immunochemical characterization of bovine brain cathepsin D. J. Neurochem. 34,284-292. Whitaker J. N. (1981) Comparative chemical, enzymatic and immunochemical features of brain cathepsin D. Comp. Biochem. Physiol. 68, 2 15-220. Whitaker J. N. and Croiwley W. R. (1983) Increased concentrations of immunoreactive cathepsin D in supraoptic nucleus of the Brattleboro rat. Br,uin Res. 277, 18 1-1 85. Whitaker J. N. and Rhodes R. H. (1983) The distribution of cathepsin D in rat tissues determined by immunocytochemistry. Am. J. Anat. 166,417-428. Whitaker J. N. and Seyer J. M. (1979) The sequential limited degradation ofbovine myelin basic protein by bovine brain cathepsin D. J. Biol. Chem. 254,6956-6963. Whitaker J. N., Terry L,. C., and Whetsell W. O., Jr. (1981) Immunocytochemical localization of cathepsin D in rat neural tissue. Brain Res. 216, 109-124. Whitaker J. N., Bertorini T. E., and Mendell J. R. (19834 Immunocytochemical stutdies of cathepsin Din human skeletal muscle. Ann. Neurol. 13, 133-142. Whitaker J. N., Dodd S. P., Sahenk Z., and Mendell J. R. (1983b) Changes in sciatic nerve cathepsin D after ligation or exposure to neurotoxins. J. llreuropathol. Exp. Neurol. 42, 87-98. Yonezawa S., Takabashi T., Wang X., Wong R. N. S., Hartsuck J. A., and Tang J. ( I 988) Structures at the proteolytic processing region ofcathepsin D. J. Biol.Chem. 263, 16504-1651 1.
Changes Induced in Astrocyte Cathepsin D by Cytokines and Leupeptin *tSJ. N. Whitaker, TP. K. Herman, tS. M. Sparacio, TS. R. Zhou, and t$E. N. Benveniste *Neurology and Research Services of the Birmingham VA Medical Center, and the Departments of ?Neurology and $Cell Biology of ihe University of Alabama at Birmingham, Birmingham, Alabama, U.S.A.
Abstract: Cathepsin D is widely, but unevenly, distributed among cells and is capable of degrading a number of neural peptides and proteins. The present study was undertaken to examine the level of cathepsin D in astrocytes that might be relevant to its induction in inflammatory demyelination. Primary astrocytes were cultured from neonatal rat cerebrums according to the method of McCarthy and de Vellis. Based on staining for cell markers, cultures were >95% astrocytes and 95% of cells present in the culture. The cathepsin D immunoreactivity varied in intensity among cells and was distributed in a granular, presumably lysosoma1 pattern. J. N e ~ i r d w r n . .VoL S 7 , No. 2, 1991
We examined whether cathepsin D levels in astrocytes could be modulated with a variety of stimuli, including cytokines. An inherent problem in the study of primary astrocytes, as compared with an astroglial cell line, is their variation among different batches. This variation was noted in astrocyte cathepsin D even after relating the immurioreactive cathepsin D level to protein. The values of 0.3-0.9 ng of cathepsin D/pg of protein are similar to results obtained on rat neural tissue specimens (Snyder and Whitaker, 1983; Whitaker and Crowley, 1983). Because of this variation, for comparisons and statistical analysis, it was necessary to standardize internally the measurements of cathepsin D changes. Thus, the measurement of cathepsin D, in nanograms per micrograms of protein, in cells grown in culture medium alone, was assigned a value of 1.O, and induced changes were expressed as a ratio (see legend of Fig. 2 for additional details). Exposure of cultured neonatal rat astrocytes to leupeptin led to a rise in cellular cathepsin D immunoreactivity (Fig. 2). Little change occurred at 16 and 24 h, but an increase of I .4to 2.0-fold from the control level of approximately 0.5 ng of cathepsin D/pg of protein could be detected by 48-72 h of culture in a dose-dependent fashion over a concentration range of leupeptin ( 1- 100 pg/ml). A concentration of 2 5 pg/ml of leupeptin was shown to be optimal for stimulating the increase in cellular levels of cathepsin D over 48 h of culture. An increase in cathepsin D immunoreactivity was clearly evident when astrocytes were cultured under serum-free conditions. Results of several studies supported the conclusion that the specific activity of astrocyte cathepsin D rose after leupeptin treatment. Total protein content did not decrease and actually increased in leupeptintreated compared with control cultures (Fig. 2A); hence, the increase in cathepsin D was due to increased cathepsin D synthesis and not a decline in cell protein. As determined by the two-way analysis of variance, the difference in the increase in cathepsin D between 16 and 48 h in the presence of leupeptin was significant ( p < 0.01). The leupeptin-inducedincrease in cathepsin D between 16 and 24 h as well as between 24 and 48 h was also significant ( p < 0.05). Repeat experiments revealed a mean (fSD) increase in the ratio, as illustrated in Fig. 2C, at 48 h of 1.7 f 0.1 (n = 3) and at 72 h of 2.1 k 0.4 (n = 7). Supernatants from cultured astrocytes revealed no change in cathepsin D immunoreactivity after treatment with leupeptin, implying no change in secretion of cathepsin D effected by leupeptin. The observed increase in cathepsin D by leupeptin was not an artifact due to any effect of leupeptin on the radioimmunoassay, in that leupeptin added directly to the immunoassay tubes at concentrations of 25- 100 pg had no effect on the assay results. Cathepsin D immunoreactivity detected in control and leupeptinexposed astrocytes demonstrated immunochemical parallelism (Whitaker et al., 198l), indicating that the cathepsin D produced in basal and leupeptin-stimulated cultures was identical immunochemically.
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FIG. 1. Cathepsin D immunofluorescenceof astrocytes. Astrocytes were plated on coverslips, cultured for 2 days, washed, and fixed for 10 s in cold acetone. The cells were stained with a polyclonalrabbit antibody (R164) to rat cathepsin D (1 :loo),then with fluoresceinated goat anti-rabbit immunoglobulin (1:20).The cells were visualized by fluorescence microscopy for total number of cells (nuclei observed with the Hoechst stain) (top) and cells immunoreactive for cathepsin D (bottom); X800.
With leupeptin as a positive control, a number of cytokines known to modulate gene expression in astrocytes (Benveniste, 1988) and other substances such as LPS, calcium ionophore, and substance P were tested for their ability to influence cathepsin D levels. At all concentrations tested and for up to 72 h of incubation, neither human recombinant TNF-a nor rat recombinant IFN-y increased cathepsin D levels compared with untreated control cells. No effect was noted when IFNy and TNF-a were added simultaneously at concentrations of 80 U/ml and 50 ng/ml, respectively, to astrocyte cultures. LPS ( 10 pg/ml), substance P ( 1- 1000 nM), IL-lp (0.1-10 ng/ml), and calcium ionophore (5 p M ) effected no change in astrocyte cathepsin D content at 48 h. As previously noted (Whitaker, 1980), the enzyme assay of cathepsin D is much less sensitive than the immunoassay. Cathepsin D enzyme activity, however, could be detected in the cultured astrocytes. Control astrocytes had enzyme activity that solubilized 5.4-
5.6%, approximately O.O4%/pgof protein, of the added substrate after culture for 48-72 h. This increased 1.2and 1.6-fold after exposure for 48 and 72 h, respectively, to leupeptin 2 5 pg/ml. The cathepsin D enzyme activity and its increase on leupeptin exposure were inhibited by pepstatin (8 pglml), indicating that the enzyme activity was in fact due to cathepsin D. Because of the increased cathepsin D appearing after exposure of rat astirocytes to leupeptin, quantitative changes in cathepsin D mRNA were studied. Northern blot analysis demonstrated that cathepsin D mRNA transcripts of the appropriate size (2.2 kb) were present in cultured astrocytes, and specifically increased by leupeptin (Fig. 3). Kinetic analysis of cathepsin D mRNA expression was examined by slot blot analysis. The mean k SD of two separate experiments is represented graphically in Fig. 4A. Cathepsin D mRNA began to increase between 12 and 16 h, reached a maximal induction of a fourfold increase over control at 24 h, and declined at 48 h. Cyclophilin mRNA levels J. Neurochem.. Val. 57, No. 2, 1991
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FIG. 3. Cathepsin D rnRNA expression in astrocytes. Northern blot analysis of cathepsin D rnRNA. Astrocytes were incubated with or without leupeptin(25 pg/ml) for 16 h. Samples (15.0 pg) of total cellular RNA isolated from unstimulatedrat astrocytes (lane A) and leupeptin-stimulatedrat astrocytes (lane B) were denatured with formaldehyde for 15 rnin at 55°C. The RNA was size-fractionatedby electrophoresis through a 1.O% agarose gel containing ethidium bromide for visualization of 28s and 18s ribosomal RNA. The RNA was then transferred to nitrocellulose in 2OX SSC. The blot was probed with cathepsin D cDNA and exposed at -70°C for 72 h to Kodak X-Omat AR film plus two intensifying screens. Note the pronounced increase in the 2.2-kb RNA species after exposure to leupeptin.
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Leupeptin (pglml) FIG. 2. Changes in cathepsin D levels in astrocytes exposed to leupeptin. A: Cells were cultured in the presence of 0-100 pg/ml of leupeptinfor 48 h at 37"C, and cell homogenates were analyzed for amounts of total protein (0)and immunoreactive cathepsin D (m). 8: The relative amount of cathepsin D (0)increasedover control at all concentrations of leupeptin tested. C Astrocytes cultured with 0-1 00 pg/ml of leupeptin for 16 ( O ) , 24 (A),and 48 (0)h at 37°C revealed increases in specific immunoreactivityof cathepsin D. Cathepsin D was quantified by radioimmunoassay, related to the amount of total protein present, and graphed as a ratio of the amount of cathepsin D (in nanograms per micrograms of protein) in leupeptin-treatedcultures to the amount of cathepsin D in cultures with no leupeptin. For results shown in (C), the control values were 0.67-0.93 ng cathepsin D/pg of protein.
were examined as a control gene as levels of this gene are generally not altered on cell stimulation (Bethea et al., 1990; Melner et al., 1990). The cathepsin D blots were stripped and reprobed for cyclophilin mRNA. There was no increase in cyclophilin mRNA at 12 or J . Nrurochem., Vol. 57, No. 2,I991
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Time (hours) FIG. 4. Kinetic analysis of cathepsin D and cyclophilin mRNA expression. A Slot blot analysis of cathepsin D mRNA. Total cellular RNA from astrocytes cultured without (0)or with leupeptin (25 pg/ml) (0) for 12, 24, and 48 h was diluted over a range of 6.01.5 pg, blotted onto nitrocellulosepaper, cross-linked,and probed with a labeled cathepsin D cDNA probe. The blot was exposed at -70°C for 24 h to Kodak X-Omat AR film plus two intensifying screens. mRNA values are expressed in arbitrary units as determined from densitometricscanning of autoradiographs.The mean f SD of two experiments is represented graphically. 8: Slot blot analysis of cyclophilin mRNA. Slot blots probed for cathepsin D mRNA (A) were stripped and reprobed with labeled cyclophilin cDNA probe. The blot was exposed at -70°C for 14 h to Kodak X-Omat AR film plus two intensifying screens. Cyclophilin mRNA from astrocytes cultured without (0)or with (0) leupeptin was expressed as for cathepsin D mRNA. The mean SD of two experiments is representedgraphically.
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48 h. An increase ( 1.5-fold) in cyclophilin mRNA was noted at 24 h on stimulation with leupeptin (Fig. 4B). Thus, both cyclophilin and cathepsin D mRNA expression are modulated by leupeptin, although cathepsin D levels are enhanced to a greater extent. The molecular features of cathepsin D in astrocytes were examined by immunoblotting of electrophoretically transferred, separated astrocyte proteins (Fig. 5). The major band of immunoreactive cathepsin D in astrocytes had a molecular mass of 40-42 kDa (Fig. 5B), identical in migration with that of cathepsin D isolated from whole rat brain (Whitaker, 1981). The dominant band of immunoreactive cathepsin D in cultured astrocytes was broad. Although this broad band could be comprised of several closely migrating proteins of similar size, it is more likely the result of the low dilution of anti-cathepsin D antiserum selected to demonstrate other bands of lower intensity. With increasing amounts of protein applied to the gels, other bands of approximately 68, 45, and 35 kDa reactive
FIG. 5. SDS-PAGE and immunoblot analysis of cathepsin D immunoreactivity in cultured primary astrocytes. Primary astrocytes (1 X 10’) were cultured in serum-free media alone (lanes 1 and 2) or with leupeptin at a concentration of 5 pg/ml (lanes 3 and 4), 25 pg/ml (lanes 5 and 6), or 125 pg/ml (lanes 7 and 8) for 48 h at 37°C.The medium was removed, the cell pellet placed in 200 pl of sample buffer, and processed for SDS-PAGE in 15% gels. Forty micrograms (lanes 1 , 3,5,and 7)or 20 pg (lanes 2, 4, 6, and 8) of protein was applied in each lane. The gel was stained with Coomassie Blue (A) or processed for electrophoretic transference of separated proteins, which were subsequently reacted with antirat cathepsin D (B). Molecular weights (X10-3) of markers are indicated. The arrow points to a 45-kDa molecular band that has been tentatively identified as procathepsin D.
with anti-cathepsin D were noted (Fig. 5B). None of these bands corresponded with the main bands detected by staining with Coomassie Blue (Fig. 5A). The pattern of cathepsin D immunoreactivity in leupeptin-treated astrocyteswas basically the same as controls except for a band, estimated at 45 kDa molecular mass, migrating just above the broad cathepsin D band that was more abundant in astrocytes treated with 25 and 125 pg/ml of leupeptin (Fig. 5B, lanes 5-8, arrow). The nature of this 45-kDa immunoreactive protein is unknown, but it has tentatively been identified as the procathepsin D. Pulse-labeling experiments will be required to validate this identification.
DISCUSSION The events and control of intracellular proteolysis in astrocytes are poorly understood but presumably involve lysosomal proteinases such as cathepsin D, which astrocytes contain in vivo (Snyder et al., 1985). The present study demonstrates that cathepsin D can be detected in cultured rat astrocytes at a concentration of 0.3-0.9 ng/pg of protein, a level approximately the same as some rat brain nuclei (Whitaker and Crowley, 1983), spinal cord (Snyder and Whitaker, 1983), and sciatic nerve (Whitaker et al., 1983a,b). Although astrocyte cathepsin D could be increased by leupeptin, cytokines such as recombinant rat IFN-y, TNF-a, a combination of IFN--/ and TNF-a, and IL- 1/3, did not increase astrocyte cathepsin D protein, nor did substance P, LPS, calcium ionophore, or LPS plus calcium ionophore. In a previous study, it was shown that cultured rat primary astrocytes, exposed to conditioned media derived from human peripheral blood mononuclear cells stimulated with either concanavalin A or phytohemagglutinin (PHA), have an increase in cathepsin D enzyme activity. PHA alone, purified human IL- 1, purified human IL-2, and recombinant human IFN-y produced no change (Bever et al., 1989). The present investigation also revealed no stimulatory effect of a variety of cytokines on astrocyte cathepsin D, implying that other types, amounts, or combinations of cytokines are responsible for the stimulatory effects observed (Bever et al., 1989).Leupeptin also increased cathepsin D mRNA levels in primary astrocytes. Whether the effect of leupeptin is exerted at the transcriptional or posttranscriptional level is as yet unknown. Thus, cathepsin D is inducible in astrocytes, but the normal or pathological factors capable of its induction in vivo remain uncertain. Leupeptin also appeared to alter cellular processing of cathepsin D in increasing the amount of cellular procathepsin D, similar to that observed in cardiac fibroblasts (Samarel et al., 1989), without increasing the amount secreted into the culture medium. Similar to other lysosomal proteinases, the mRNA of cathepsin D leads to the synthesis of a preprocathepsin D that loses its 20 residue signal peptide and leaves the Golgi apparatus as the glycoprotein procathepsin D (EnckJ. Neurorhem., Vol. 57, No. 2. 1991
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son, 1989). Procathepsin D, believed to be inactive as an enzyme, loses its 44-residue propiece en route to or in the lysosome. Its single chain has a molecular mass of 40-45 kDa and may be cleaved further, in a speciesdependent fashion, to a heavy chain of 30 kDa and a light chain of 15 kDa (Erickson, 1989). Rats have mainly the single chain form of cathepsin D (Whitaker, 1981), but may have a minor portion of the two-chain variety (Yonezawa et al., 1988). The mechanism for the conversion of procathepsin D to cathepsin D is uncertain, but has been postulated to be a result of leupeptin-sensitive proteinases (Hentze et al., 1984; Gieselmann et al., 1985) or of autocatalysis (Conner, 1989). In the synthesis of cathepsin D, the proform may have a molecular mass of 46-52 kDa (Hasilik and Neufeld, 1980; Westley and May, 1987). Higher molecular weight bands of immunoreactive cathepsin D of different sizes have been noted in human promonocytes (Stein et al., 1987), human skin fibroblasts (Hasilik and Neufeld, 1980), and C6 glioma cells (Snyder and Whitaker, 1988). Their identity and relationship to the other molecular species of cathepsin D are unknown. Similar to the primary astrocytes studied in this investigation, cathepsin D is present in C6 glioma cells (Snyder and Whitaker, 1988). Cathepsin D in C6 glioma cells appears in different sizes besides the dominant 42-kDa form and can be modified by cyanate and monensin presumably through an effect on the Golgi apparatus (Snyder and Whitaker, 1988). The cDNA for cathepsin D has been obtained from a human hepatoma library (Faust et al., 1985) and by sequencing an mRNA induced in a human breast cancer cell line, MCF7, by estradiol (Westley and May, 1987). The cathepsin D gene has been localized to the pl5 band of chromosome 1 1 in the MCF7 breast cancer cell line (Augereau et al., 1988). The mRNA of cathepsin D has a size of 2.1-2.2 kb and contains sequences for the amino terminal pre- and pro-portions of the proteinase (Faust et al., 1985). The size of the cathepsin D mRNA in astrocytes reported in the present investigation is the same (2.2 kb). Although cathepsin D mRNA seems to be rather stable in cultured cells (Faust et al., 1985), it may be induced in selected cells by estradiol, epidermal growth factor (Cavailles et al., 1989), and calcitrol (Redecker et al., 1989). Cathepsin D mRNA levels are relatively uniform in different rat tissues (San Segundo et al., 1986), even though cathepsin D localization may vary widely among different cells of rat organs (Whitaker and Rhodes, 1983). Cathepsin D mRNA showed no changes in a myogenic rat cell line cultured in the presence of serum (Colella et al., 1986). Leupeptin is a naturally occurring, water-soluble proteinase inhibitor derived from streptomyces (Umezawa, 1976). Leupeptin is small, having a molecular weight of 475, binds well to trypsin and cysteine proteinases, and is effective as an inhibitor for many proteinases including plasmin, trypsin, papain, calpain (Umezawa, 1976;Vitto and Nixon, 1986),the cysteine
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proteinases including cathepsins B, H, and L (Umezawa, 1976), and the trypsin-like site on multicatalytic proteinase (Rivett, 1989). Leupeptin has an effect, usually presumed to be a result of its properties as a proteolytic inhibitor, on several cell functions. These include a reduction of the PHA-induced proliferation of guinea pig lymphocytes (Saito et al., 1972), inhibition of the growth of normal and polyoma-transformed baby hamster kidney cells (McIlhinney and Hogan, 1974), and severe growth retardation and abnormal development of rat conceptuses in vitro (Freeman and Brown, 1985). Cerebral intraventricular injection of leupeptin can block olfactory discriminitive learning interpreted to be the result of inhibition of proteinases involved in memory formation (Staubli et al., 1985). Leupeptin may have biological properties other than those of a proteolytic inhibitor. Leupeptin added to primary cultures of adult rat hepatocytes predictably diminished cathepsin D activity but unexpectedly led to an induction of an acid proteinase, partially inhibited by pepstatin (Tanaka et al., 1979). Similar to our findings with cultured primary rat astrocytes, cathepsin D activity was maximally stimulated in rat hepatocytes by 25-50 pg/ml of leupeptin. Although the detection was by enzymatic activity rather than by immunoassay, the leupeptin-stimulated rat hepatocyte responded more quickly and to a greater extent than did the cultured astrocytes. The induction of the acid proteinase activity by leupeplin was not a property of all small peptide inhibitors of microbial origin (Tanaka et al., 1981) and could be prevented by inhibition of RNA and protein synthesis (Tanaka et al., 1979). The exact nature of the acid proteinase induced by leupeptin in rat hepatocytes was not delineated; however, it was later shown to have features of a cysteine proteinase similar to, but not identical with, cathepsin L (Tanaka et al., 1981;Tanaka et al., 1984). The present results provide proof at the RNA, protein, and enzyme levels that the proteinase induced by leupeptin in astrocytes is cathepsin D. The cathepsin D mRNA was increased between 12 and 16 h, followed by a cathepsin D immunoreactivity increase at 24-48 h. The fact that leupeptin induced cathepsin D and another proteinase indicates that some of the cellular changes attributed to leupeptin on the basis of its property as a proteinase inhibitor may be due in part to its effect on inducing proteinases and, possibly, other cellular constituents. The role of cathepsin D and other proteinases in the variety of functions attributed to astrocytes remains to be defined. Some naturally occurring proteinase inhibitors, especially a,-antitrypsin, a2-macroglobulin, and antithrombin 111, stimulate astrocyte proliferation and a small increase in1 glutamine synthetase (Perraud et al., 1988). Astrocyles may serve as antigen-presenting cells, a function that requires proteolysis, if first induced by IFN-7 (Fontana et al., 1984), virus (Massa et al., 1986),or I F N y plus TNF-a (Benveniste et al., 19898), to express major hlistocompatibility complex class I1 antigens. This is suggested as the principal way in which
CHANGES INDUCED IN CATHEPSIN D
astrocytes might initiate or support an in situ immune response in inflammatory demyelination (Fontana et aI., 1987). Cathepsin D is a likely participant in antigen processing (Diment and Stahl, 1985; van Noort and van der Drift, 1989). As shown by the present study, one of the conditions for induction of major histocompatibility complex class 11, that is, IFN-./ and TNF-a stimulation, did not lead to an increase in astrocyte cathepsin D. The actions of cathepsin D or the quantitative levels needed in the antigen-processing function of astrocytes are unknown. The induction of astrocyte cathepsin D presumably requires biological factors, alone or in combination, that are not yet identified. Acknowledgment: This research was supported by the Research Program of the Veterans Administration (J.N.W.) and by National Institutes of Health Grant A1 27290 (E.N.B.). We thank Mr. Alan Clayton for technical assistance, Mr. Albert Tousson for assistance in fluorescence microscopy, Mr. Feng Sun for assistance in the statistical analysis, and Ms. Joanne Cage for assistance in preparation of the manuscript.
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