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Calmodulin regulates the interleukin 1-induced procollagenase production in human uterine cervical fibroblasts
61
Biochimica et Biophysica Acta, 1011 (1989) 61-66 Elsevier
BBA 12430
Calmodulin regulates the interleukin 1-induced procollagenase production in human uterine cervical fibroblasts Yuuki Ojima 1, Akira Ito 1, Hideaki Nagase 2 and Yo Moil l t Department of Bioct,emist~, Tokyo College of Pharmacy, Horinouchi, Hachioji, Tol~vo(Japan) and h Department of Biochemistry, University of Kansas Medical Center, Kansas City, KS (U.S.A.)
(Received 27 June 1988) (Revised manuscript received 15 November 1988)
Key words: Calmodulin; Recombinant interleukin la; Procollagenase; W-7; Human cervix; (Fibroblast)
Interleukin I (IL-I) stimulates the synthesis of collagenase in human uterine cervical fibroblasts. This inductive effect of IL-I on collagenase production was augmented by N-(6-aminohexyi)-5-chloro-l-naphthalenesulfo~amide (W.7), a specific inhibitor off calmodulin, in a dose-dependent manner. The apparent co|lagenase activity observed in tile cuhurc medium of the cells treated with |L-| and 40/tM W-7 was about three times higher than that produced by the cells treated with |L-I alone. The immunoblotting with the specific antibody against human coilagenase showed that the increased collagenase activity resulted from the accelerated biosynthesis of collagenase. Another calmodulin inhibitor, trifluoperazine, enhance( the effect o[ |L-I on collagenase production similarly. However, the effect of N-(6-aminohexyl)-l-naphthalenesulfonamide, the weakest inhibitor o| caimodulin, wa~ negligible. These results suggesl that W-7 enhances the collagenase production by specifically inhibiting calmodulin and that calmodulin may act as a suppressor o[ the |L-l-induced collagenase production in human uterine ceivical fibroblasts.
introduction Interleukin 1 (IL-1), a cytokine typically produced by monocytes/macrophages, exerts a number of biological activities both in vivo and in vitro (see Ref. 1 for a review). In addition to its crucial role in lymphocyte proliferation, IL-1 has been shown to be one of the inflammatory mediators [1] and to participate in the induction of fever and acute-phase protein [2,3], and connective tissue matrix catabolism [4]. The degradation of extracellulat matrix components induced by IL-1 is thought to be due to the accelerated production of proteolytic enzymes by connective tissue cells. IL-I stimulates synovial fibroblasts or articular chondrocytes to produce collagenase, some other metalloproteinases
Abbreviations: IL-I, interleukin 1; W-7, N-(6-aminohexyl)-5-chloro1-naphthalenesuifonamide; W-5, N-(6-aminohexyl)-l-naphthalenesulfonamide; TFP, trifluoperazine; MCM, macrophage-conditioned medium; MEM, Eagle's minimum essential medium; FCS, fetal calf serum; LAH, lactalbumin hydrolysate; SDS-PAGE, sodium dodecyi sulfate-polyacrylamide gel electrophoresis. Correspondence: A. lto, Department of Biochemistry, Tokyo College of Pharmacy, Horinouchi, Hachioji, Tokyo 192-03, Japan.
and prostaglandin E2 [5-71. We have reported that the uterine cervices of pregnant rabbits produce IL-l-like factors, which stimulate the production of collagenase in rabbit uterine cervical fibroblasts [8,9]. However, the mechanism of the action of IL-I underlying these responses is not clear. Recently, cell surface IL-1 receptors have been identified in various connective tissue cells [10,11]. More recently, it has been reported that recombinant human IL-lfl stimulates procollagenase gene expression and accumulates cellular procollagenase mRNA in human articular chondrocytes [12]. These observations suggest that the induction of collagenase production by I L-1 is regulated at the transcriptional level in connective tissue cells, and that it is mediated via its cell surface receptors, but the intracellular mediators of IL-1 action have not been clarified. In investigating some of the mechanisms which regulates collagenase production, Dayer et al. [13] reported that a calmodulin inhibitor, trifluoperazine (TFP), potentiates the effect of a mononuclear cell factor(s) on the production of collagenase by human synovia! cells. Although TFP binds to the CaZ+-calmodulin complex, it is not clear whether the augmenting effect of TFP on the collagenase response to the mononuclear cell factor is due to the inhibition of calmodulin activity or to its
0167-4889/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
62 lipophilic effects on cell membranes. Relatively high hydrophobicity of TFP is recognized [14,151. In this report, therefore, using a less non-specific calmodulin inhibitor, W-7, we investigated the role of calmodulin in collagenase production by the human uterine cervical fibroblasts treated with II.-1. The compound, W-7, was found to be water soluble, and capable of penetrating the cell membrane [16,17]. The stimulatory effect of W-7 on the collagenase induction by IL-1 suggests that calmodulin is a suppressor of the effect of IL-1 on collagenase production in human uterine cervical fibroblasts. Materials and Methods
Materials W.7 and W-5 were purchased from Seikagaku Kogyo, Co., Ltd, Tokyo, Japan. TFP, alkaline phosphatase-conjugated donkey anti-(sheep IgG)IgG, 5-bromo-4-chloro3-indolyl phosphate and Nitro blue tetrazolium were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. Human recombinant IL-la (2-10 7 units/mg) was kindly supplied by Dainippon Pharmaceutical Co., Suita, Osaka, Japan. Other reagents used were of analytical grade.
Collagenase assay Trypsin-activatable collagenase activity was measured using [~4C]acetylated, reconstituted fibrils of guinea pig skin collagen according to the method of Cawston and Barrett [20] with slight modifications as described previously [19,21]. 1 unit of collagenase degrades 1 pg of collagen per min at 37" C. Determination of cellular DNA content The content of cellular DNA was determined by a fluorescence DNA assay using 3,5-diaminobenzoic acid according to the method of Wint and Hollis [22]. Western blotting Procollagenase was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 12.5% acrylamide slab gels [23] under reducing conditions. The samples were first subjected to SDS-PAGE and then electrotransferred to a nitrocellulose filter. The filter was reacted with sheep anti-(human synovial procollagenase) antibody which was then complexed with alkaline phosphatase-conjugated donkey anti-(sheep IgG)IgG. Immunoreactive collagenase was visualized indirectly using 5-bromo-4-chloro-3-indolyl phosphate and Nitro blue tetrazolium. Results
Preparation of macrophage.conditioned medium Peritoneal macrophages were obtained from female rabbits (Nippon white) by intraperitoneal injection of 0.2% (w/v) glycogen, and the conditioned medium was prepared by costimulation of these cells with lipopolysaccharide as described previously [18]. Gel permeation chromatography indicated that there are three species with different molecular weights, all of which contain both the stimulatory effects on collagenase production and lymphocyte proliferation [9]. Cell culture and treatment Uterine cervices from women of child-bearing age were obtained from patients undergoing hysterectomy of fibromyoma not involving the cervix. The culture of stromal fibroblasts was established as described for cells from rabbit uterine cervix [19]. The cells were plated at a density of 1.10 s cells/ml of 10% (v/v) FCS/MEM in a 24 multiwell plate, and grown to confluence. In most experiments, cells with up to the fifth passage were used. After confluence, the collagenase production was ~nduced for 6 days by adding 50% (v/v) macrophageconditioned medium (MCM) or human recombinant IL-la (10 ng/ml) in the serum-free medium containing 0.2% (w/v) lactalbumin hydrolysate (LAH). Then the medium was replaced by W-7 and/or IL-l-supplemented medium and the culture was maintained for suitable periods.
Sti nulation of the IL-l-induced collagenase activity by co, :wd'.:!!,~ ,lntagonists Human uterine cervical fibroblasts in the culture produced no significant amount of collagenase, but the production of collagenase was stimulated by treating the cells with MCM or recombinant IL-la. Thus, to maintai,~ a constant level of collagenase production, .,'onfluent cells were first treated with 50% (v/v) MCM twice cor.secutively for 6 days, and they were then used for subsequent experiments. To e,~amine the effect of W-7 on MCM-induced collagenase production, fibroblasts primed with 50% (v/v) MCM were exposed to the reagent for 24 h in the presence of MCM. As shown in Fig. 1, the apparent collagenase activity detected in the culture media was stimulated by W-7 in a dose-dependent manner over the concentration range of 5-40 /~M. About 3-fold enhancement of collagenase production above the basal level was detected with 40 pM W-7. However, this enhancing effect of W-7 was not observed under the absence of IL-la (see below in Table I, Expt. 1) or MCM (data not shown). To confirm further the fact that the enhancing effect of W-7 on the MCM-induced procollagenase production is due to the inhibition of calmodulin, the effects of W-5 and TFP on the enzyme production were examined. W-5, a chloride-deficient analogue of W-7 that binds to calmodulin only weakly [17], showed no significant
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W-7orW-5 Concentration (~M) Fig. 1, The dose-dependent effect of W-7 or W-5 on the MCM-induced collagenase activity in human uterine cervical fibroblasts. Cervical fibroblasts at the third passage were maintained in 10% Iv/v) F C S / M E M until confluence, and then the culture medium was changed to 0.2% ( w / v ) L A H / M E M . The confluent cultures were first incubated with 50% (v/v) MCM as described in Materials and Methods. After 6 days, the cells were treated with W-7 or W-5 at the concentrations indicated (5-40 #M) for 24 h in the presence of 50,~ (v/v) MCM. The culture media were harvested for collagenase assay; o , W-7; ®o W-5. Each point represents the mean _+S.D. of three wells.
TABLE I
Effect of W-7 on the recombinant human IL-la- or MCM-dependent collagenase production by human uterine cervical fibroblasts Confluent cervical fibroblasts at primary (Expt. 1) or third passage (Expt. 2) were preincubated twice with 10 n g / m l recombinant human I L - l a or 50% ( v / v ) MCM for 6 days. Then the cells were treated with 40 laM W-7, 10 n g / m l I L - l a (or 50% (v/v) MCM) or W-7 plus IL-la (or MCM) in 0.2% ( w / v ) L A H / M E M . After 24 h, the culture media were harvested and assayed for collagenase activity, and then the cells were washed twice with phosphate-buffered saline and the cellular DNA content was determined as described in the text. Data are mean values_+ S.D. of three wells, n.d., not detectable. Treatment
Collagenase activity (units/ml)
Cellular DNA content (/t g/well)
Expt. 1 None W-7 (40/tM) It-1 a (10 n g / m l ) IL-la +W-7
n.d. n.d. 1.29 + 0.07 3.25+0.24 "
18.46 + 0.37 19.68 + 0.20 19.99 + 0.26 18.87+0.08
Expt. 2 None MCM (50% (v, v)) MCM +W-7 (40 ~M)
n.d. 2.24 __0.26 7.43_+1.29 b
16.25 + 0.15 17.80 _+0.24 17.55_+0.11
" Significant difference, P < 0.05, compared to corresponding value in IL-la. b Significant diff6;cnce, P < 0.Ol, compared to corresponding value in MCM.
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TFP Concentration (jiM) Fig. 2. Effect of T F P on MCM-induced collagenase production by Imman uterine cervical fibroblasts. Cervical fibroblasts at the third passage were incubated with 50f~ (v/v) MCM as described in Fig. !. Then the cells were exposed to various concentrations of TFP in the presence of 50e~ (v/v) MCM for 24 h. Each point repre.~cnts the mean + S.D. of three wclls.
stimulative effect on the coilagenase response to MCM (Fig. 1). In contrast, TFP, a calmodulin inhibitor of phenothiazine derivative [24], effectively promoted the MCM-induced collagenase activity to a similar extent as W-7 (Fig. 2). Although TFP has a relatively high hydrophobicity [14,15], and the nonspecific effects of TFP on cell membrane are not ruled out, the facts that W-7, a more specific plasma membrane-permeable calmodulin inhibitor, but not W-5 enhanced MCM-induced collagenase production and that TFP also stimulated the MCM-induced collagenase to a similar extent, suggest that calmodulin prevents the collagenase gene from being fully activated by MCM. A similar effect of W-7, but to a lesser extent, was also seen with cells treated with human recombinant IL-la (Table l), indicating that It-1 in MCM is a leading candidate ligand which induces collagenase. On the other hand, W-7 did not affect the cellular DNA content (Table I) and the cell number (data net show~). Therefore, the enhanced production of collagenase induced by W-7 was not due to the proliferation of ceils or the cytotoxic effect of the reagent. However, W-7 was not effective at concentrations higher than 50/.tM. At these concentrations, W-7 was found to be toxic to cervical fibroblasts: a considerable decrease in both cell number and cellular DNA content were observed (data not shown).
Effecl of W-7 on the production of procoilagenase It is well recognized that connective tissue cells in culture synthesize an endogenous metalloproteinase inhibitor (tissue inhibitor of metalloproteinases: TIMP) as
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Fig. 3. Enhanced production of procollagenase by W-7 from human uterine cervical fibroblasts. Confluent fibroblasts at the fourth passage were treated with 50% (v/v) MCM as described in Fig. 1. After 6 days, the cells were treated or not with 50% (v/v) MCM, or 40/,M W-7 plus MCM for 24 h. The harvested crude culture media (0.5 ml) were mixed with 0.25 mi of 10~ (w/v) trichloroacetic acid. The precipitates were collected by centrifugation, dissolved in 15 /,I of reducing sample buffer and subjected to SDS-PAGE. The procollagenase bands were visualized by immunoblotting as described in Materials and Methods, Lane !, no treatment: lane 2, MCM; lane 3, MCM plus W-7.
well as collagenase. Therefore, we examined that the enhanced collagenase activity found with W-7/MCM or IL-l-treated cells is due to the increased biosynthesis of the collagenase molecule. The amount of collagenase produ~;don wa~ measured by immunoblotting, using the specific antibody raised against human synovial collagnease. In the control culture, procollagenase was not detected immunologically (Fig. 3, lane 1) as predicted from the measurement of collagenase activity. When cervical fibroblasts were treated with MCM, procollagenase was obviously induced and detected as two species with Mr values of 55000 and 57000 (lane 2), and the production of procollagenase was augmented by the co-treatment of cells with W-7 (lane 3). This stimulative effect of W-7 on the procollagenase production was inhibited almost completely by 1 . 1 0 -5 M cycloheximide. Furthermore, W-7 did not cause any increase in the synthesis of the total cell proteins by human cervical fibroblasts (data not shown). Thus, the action of W-7 towards procollagenase production was
Fig. 4. Effect of W-7 and W-5 on the time course of the MCM-induced collagenase production by human uterine cervical fibroblasts. Cervical cells at the fifth passage were grown in 10% (v/v) FCS/MEM to confluence. Then the cells without MCM priming were treated with the following reagents in serum free MEM containing 0.2% (w/v) LAH and the media were changed daily for 6 days; o, W-7 (10/,M) and MCM (50% (v/v)); e, W-5 (10/,M) and MCM (50% (v/v)); A, MCM (50% (v/v)). Data are shown as the mean + S.D. of three wells.
concluded to be due to the direct modulation of the enzyme biosynthesis.
Effect of W-7 on the time course study of collagenase production T h e effect o f W - 7 o n I L - l - i n d u c e d c o ! ! 2 g e n a s e p r o d u c t i o n in cervical f i b r o b l a s t s was e x a m i n e d w i t h o u t
o I
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Days in culture Fig. 5. Influence of W-7 priming on the MCM-induced collagenase production in human uterine cervical fibroblasts. Confluent cervical cells at the second passage without MCM pretreatment were treated with W-7 in 0.2% (w/v) LAH/MEM for 24 h. Then the cells were maintained in 0.2% (w/v) LAH/MEM containing 50% (v/v) MCM, all the media were changed daily for 4 days. The abscissa indicates the days of culture after the removal of W-7. 4, 10 ~M W-7 primed; [2, 20 /tM, o, 40 ~M; A, non-primed control. Data are shown as the mean + S.D. of three wells.
65 MCM priming. Fig. 4 shows the time-dependent increase in collagenase production by 10 pM W-7 in the presence of 5070 (v/v) MCM. The same concentration of W-5, however, failed to enhance the collagenase production. These results indicate that the effect of W-7 in human cervical fibroblasts is also expressed by the consecutive treatment at a relatively low concentration of W-7 (10 #M), and is maintained when the cells coexists with W-7. On the other hand, in the absence of MCM, the cells produced no significant collagenase activity even after consecutive treatment with W-7 (10 #M) (data not shown). The responsiveness of the cervical cells pretreated with W-7 to MCM was also examined. The results are shown in Fig. 5. When the cells were primed with W-7 for 24 h, the inductive effect of MCM on collagenase production appeared more rapidly, in a dose-dependent manner, as compared with the cells with MCM alone. The enhancing effect of W-7 diminished 3 days after priming, indicating that this effect is reversible. Discussion
In the present study, we have demonstrated that the inhibition of calmodulin and the increase in the IL-l-induced collagenase production appear to be correlated. However, the exact roles of calmodulin on the increase in IL-l-induced collagenase are still unknown. We consider that there are at least three possibilities which could account for the above situation; calmodulin acts as (i) a mediator or regulator for initial signals of IL-1, (ii) a regulator for the collagenase gene expression and (iii) a regulator for the secretion of collagenase. If calmodulin is the intracellular mediator of IL-1, there might be a good correlation between IL-1 and the level of intracellular calcium after exposure to IL-I. We did not measure the cellular calcium concentration in human uterine cervical fibroblasts. However, Matsushima et al. [25] have reported an interesting observation that in murine thymocytes, calcium ionophore A23187 synergistically enhanced the thymocyte proliferation induced by IL-1, but A23187 alone caused no thymocyte proliferation. They have suggested in a recent review [1] that the expression of biological activity of IL-1 depends on the intracellular calcium concentration, but that it does not directly alter the intracellular level of calcium. Furthermore, Georgilis et al. [26] have reported that recombinant human IL-lfl had no effect on the concentration of intracellular free calcium in human neutrophils. In view of these observations and the fact that W-7 alone did not induce collagenase production in cervical cells (data not shown) suggest that it is unlikely that the effect of IL-1 on collagenase production in uterine cervical cells is due to the changes in the intracellular calcium level, and that calmodulin is an intracellular mediator of IL-1. On the other hand, IL-1
caused a slight transient fall in the total intracellular calcium level in a murine B-cell line [27]. Thus, the precise relationship between IL-1 and intracellul:~r calcium is not yet fully understood. It is also unlikely that calmodulin negatively regulates the secretion of collagenase from cervical cells, since the rate of the collagenase release into the extracellular space is reflected predominantly by its de novo synthesis [28,29]. Furthermore, our results show that pretreatment of the cells with W-7 can rapidly augment MCM-induced collagenase production (Fig. 5). Therefore, we censider the possibility that calmodulin may act by modulating collagenase gene expression rather than the secretion of collagenase. However, it is unclear how calmodulin regulates the production of collagenase. In this respect, the fact that caimodulin affects the cytoskeleton in various types ot cell [30] is of interest. In fact, Aggeler et al. [31] have reported that various agents which affect the cytoskeleton such as cytochalasin B, colchicin and TFP induced collagenase production in rabbit synovial fibroblasts by causing some changes in the cellular shape. These observations suggest that, in human uterine cervical cells, both TFP and W-7 may alter the interaction of ,he cytoskeleton with plasma membrane by binding to calmodulin, tbus resalting in the augmentation of the IL-l-induced collagenase production. However, these authors reported in their recent paper [32] that cycloheximide effectively inhibited the ocllagenase production, but it did not prevent these morphological changes induced by cytochalasin B. Furthermore, in human uterine cervical fibroblasts, the changes in cellular shape are not essential for the enhancement of IL-l-induced collagenase production rdata not shown). In conclusion, we have demonstrated that W-7, a specific calmodulin inhibitor, stimulates the IL-l-induced collagenase production in human uterine cervical fibroblasts. Although the precise mechanism is s61l unclear, we propose that calmodulin acts as a suppressor of collagenase production in these cells. Recently, a similar suppressive effect of calmodulin on the protein synthesis has also been reported for the synthesis of the low-density lipoprotein receptor in human skin fibroblasts [33]. Acknowledgment This study was supported in part by a National Institutes of Health Grant AR39189. Re|erences 10ppenheim. J.J., Kovacs, E.J., Matsushima. K. and Durum. S.K. (1986) lmmunol. Today 7, 45-56. 2 Dinarello.C.A. and Wolff, S.M. (li982) Am. J. Med. 72. 799-819. 3 Dinarello,C.A. (1984) Rev. Infect. Dis. 6, 51-95.
66 4 McCroskery, P.A., Arai, S., Amento, E.P. and Krane, S.M. (1985) FEBS Lett. 191, 7-12. 5 Mizel, S.B., Dayer, J.M., Krane, S.M. and Mergenhagen, S.E. (1981) Proc. Natl. Acad. Sci. USA 78, 2474-2477. 6 Saklatvala, J., Pilsworth, L.M.C., Sarsfield, S.J., Gavriloric, J. and Heath, J.K. (1984) Biochem. J. 224, 461-466. 7 Gowen, M., Wood, D.D., lhrie, E.J., Meats, J.E. and Russell, R.G.G. (1984) Biochim. Biophys. Acta 797, 186-193. 8 lto, A., Hiro, D., Sakyo, K. and Moil, Y. (1987) Biol. Reprod. 37, 511-517. 9 lto, A., Hiro, D., Ojima, Y. and Moil, Y. (1988) Am. J. Obstet. Gynecol. 159, 261-265. 10 Bird, T.A. and Saklatvala, J. (1986) Nature 324, 263-266. 11 Dower, S.K., Kronheim, S.R., Hopp, T.P., Cantrell, M., Deeley, M., GiUis, S., Henney, C.S. and Urdal, D.L. (1986) Nature 324, 266-268. 12 Steph~nson, M.L., Goldring, M.B., Birkhead, J.R., Krane, S.M. Rahmsdorf, H.J. and Angel, P. (1987) Biochem. Biophys. Res. Commun. 144, 583-590. 13 Dayer, J.M., Roelke, M.S. and Krane, S.M. (1984) Biochem. Pharmacol. 33, 2893-2899. 14 Roufogalis, B.D. (1981) Biochem. Biophys. Res. Commun. 98, 607-613. 15 Weiss, B., Prozialeck, W.C. and Wallace, T.L. (1982) Biochem. Pharmacol. 31, 2217-2226. 16 Hidaka, H., Yamaki. T., Naka, M., Tanaka, T., Hayashi, H. and Kobayashi, R. (1980) Mol. Pharmacol. 17, 66-72.
17 Hidaka, H., Sasaki, Y., Tanaka, T., Endo, T., Ohno, S., Fujii, Y. and Nagata, T. (1981) Proc. Natl. Acad. Sci. USA 78, 4354-4357. 18 Hiro, D., Ito, A., Matsuta, K. and Moil, Y. (1986) Biochem. Biophys. Res. Commun. 140, 715-722. 19 Sakyo, K., Ito, A. and Moil, Y. (1986) J. Pharmacobio-Dyn. 9, 276-286. 20 Cawston, T.E. and Barrett, A.J. (1979) Anal. Biochem. 99, 340-345. 21 Sakyo, K., lto, A. and Mori, Y. (1987) Biol. Reprod. 36, 277-281. 22 Wint, B.J. and Hollis, S. (1982) Anat. Biochem. 122, 338-344. 23 Laemmli, U.K. (1970) Nature 227, 685-690. 24 Levin, R.M. and Weiss, B. (1977) Mol. Pharmacol. 13, 690-697. 25 Matsushima, K. and Oppenheim, J.J. (1985) Cell. Immunol. 90, 226-233. 26 Georgilis, K., Schaefer, C., DinareUo, C.A. and Klempner, M.S. (1987) J. lmmunol. 138, 3403-3407. 27 Stanton, T.H., Maynard, M. and Bomsztyk, K. (1985) J. Biol. Chem. 261, 5699-5701. 28 Vaile, KJ. and Bauer, E.A. (1979)J. Biol. Chem. 254, 10115-10122. 29 Nagase, H., Bilnckerhoff, C.E., Vater, C.A. and Harris, E.D., Jr. (1983) Biochem. J. 214, 281-288. 30 Kakiuchi, S. and Sobue, K. (1983) Trends Biochem. Sci. 8, 59-62. 31 Aggeler, J., Frisch, S.M. and Werb, Z. (1984) J. Cell Biol. 98, 1662-1671. 32 Frisch, S.M., Clark, E.J. and Werb, Z. (1987) Proc. Natl. Acad. Sci. USA 84, 2600-2604. 33 Filipovic, l. and Buddecke, E. (1986) Biochim. Biophys. Acta 876, 124-132.
Biochimica et Biophysica Acta, 1011 (1989) 61-66 Elsevier
BBA 12430
Calmodulin regulates the interleukin 1-induced procollagenase production in human uterine cervical fibroblasts Yuuki Ojima 1, Akira Ito 1, Hideaki Nagase 2 and Yo Moil l t Department of Bioct,emist~, Tokyo College of Pharmacy, Horinouchi, Hachioji, Tol~vo(Japan) and h Department of Biochemistry, University of Kansas Medical Center, Kansas City, KS (U.S.A.)
(Received 27 June 1988) (Revised manuscript received 15 November 1988)
Key words: Calmodulin; Recombinant interleukin la; Procollagenase; W-7; Human cervix; (Fibroblast)
Interleukin I (IL-I) stimulates the synthesis of collagenase in human uterine cervical fibroblasts. This inductive effect of IL-I on collagenase production was augmented by N-(6-aminohexyi)-5-chloro-l-naphthalenesulfo~amide (W.7), a specific inhibitor off calmodulin, in a dose-dependent manner. The apparent co|lagenase activity observed in tile cuhurc medium of the cells treated with |L-| and 40/tM W-7 was about three times higher than that produced by the cells treated with |L-I alone. The immunoblotting with the specific antibody against human coilagenase showed that the increased collagenase activity resulted from the accelerated biosynthesis of collagenase. Another calmodulin inhibitor, trifluoperazine, enhance( the effect o[ |L-I on collagenase production similarly. However, the effect of N-(6-aminohexyl)-l-naphthalenesulfonamide, the weakest inhibitor o| caimodulin, wa~ negligible. These results suggesl that W-7 enhances the collagenase production by specifically inhibiting calmodulin and that calmodulin may act as a suppressor o[ the |L-l-induced collagenase production in human uterine ceivical fibroblasts.
introduction Interleukin 1 (IL-1), a cytokine typically produced by monocytes/macrophages, exerts a number of biological activities both in vivo and in vitro (see Ref. 1 for a review). In addition to its crucial role in lymphocyte proliferation, IL-1 has been shown to be one of the inflammatory mediators [1] and to participate in the induction of fever and acute-phase protein [2,3], and connective tissue matrix catabolism [4]. The degradation of extracellulat matrix components induced by IL-1 is thought to be due to the accelerated production of proteolytic enzymes by connective tissue cells. IL-I stimulates synovial fibroblasts or articular chondrocytes to produce collagenase, some other metalloproteinases
Abbreviations: IL-I, interleukin 1; W-7, N-(6-aminohexyl)-5-chloro1-naphthalenesuifonamide; W-5, N-(6-aminohexyl)-l-naphthalenesulfonamide; TFP, trifluoperazine; MCM, macrophage-conditioned medium; MEM, Eagle's minimum essential medium; FCS, fetal calf serum; LAH, lactalbumin hydrolysate; SDS-PAGE, sodium dodecyi sulfate-polyacrylamide gel electrophoresis. Correspondence: A. lto, Department of Biochemistry, Tokyo College of Pharmacy, Horinouchi, Hachioji, Tokyo 192-03, Japan.
and prostaglandin E2 [5-71. We have reported that the uterine cervices of pregnant rabbits produce IL-l-like factors, which stimulate the production of collagenase in rabbit uterine cervical fibroblasts [8,9]. However, the mechanism of the action of IL-I underlying these responses is not clear. Recently, cell surface IL-1 receptors have been identified in various connective tissue cells [10,11]. More recently, it has been reported that recombinant human IL-lfl stimulates procollagenase gene expression and accumulates cellular procollagenase mRNA in human articular chondrocytes [12]. These observations suggest that the induction of collagenase production by I L-1 is regulated at the transcriptional level in connective tissue cells, and that it is mediated via its cell surface receptors, but the intracellular mediators of IL-1 action have not been clarified. In investigating some of the mechanisms which regulates collagenase production, Dayer et al. [13] reported that a calmodulin inhibitor, trifluoperazine (TFP), potentiates the effect of a mononuclear cell factor(s) on the production of collagenase by human synovia! cells. Although TFP binds to the CaZ+-calmodulin complex, it is not clear whether the augmenting effect of TFP on the collagenase response to the mononuclear cell factor is due to the inhibition of calmodulin activity or to its
0167-4889/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
62 lipophilic effects on cell membranes. Relatively high hydrophobicity of TFP is recognized [14,151. In this report, therefore, using a less non-specific calmodulin inhibitor, W-7, we investigated the role of calmodulin in collagenase production by the human uterine cervical fibroblasts treated with II.-1. The compound, W-7, was found to be water soluble, and capable of penetrating the cell membrane [16,17]. The stimulatory effect of W-7 on the collagenase induction by IL-1 suggests that calmodulin is a suppressor of the effect of IL-1 on collagenase production in human uterine cervical fibroblasts. Materials and Methods
Materials W.7 and W-5 were purchased from Seikagaku Kogyo, Co., Ltd, Tokyo, Japan. TFP, alkaline phosphatase-conjugated donkey anti-(sheep IgG)IgG, 5-bromo-4-chloro3-indolyl phosphate and Nitro blue tetrazolium were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. Human recombinant IL-la (2-10 7 units/mg) was kindly supplied by Dainippon Pharmaceutical Co., Suita, Osaka, Japan. Other reagents used were of analytical grade.
Collagenase assay Trypsin-activatable collagenase activity was measured using [~4C]acetylated, reconstituted fibrils of guinea pig skin collagen according to the method of Cawston and Barrett [20] with slight modifications as described previously [19,21]. 1 unit of collagenase degrades 1 pg of collagen per min at 37" C. Determination of cellular DNA content The content of cellular DNA was determined by a fluorescence DNA assay using 3,5-diaminobenzoic acid according to the method of Wint and Hollis [22]. Western blotting Procollagenase was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 12.5% acrylamide slab gels [23] under reducing conditions. The samples were first subjected to SDS-PAGE and then electrotransferred to a nitrocellulose filter. The filter was reacted with sheep anti-(human synovial procollagenase) antibody which was then complexed with alkaline phosphatase-conjugated donkey anti-(sheep IgG)IgG. Immunoreactive collagenase was visualized indirectly using 5-bromo-4-chloro-3-indolyl phosphate and Nitro blue tetrazolium. Results
Preparation of macrophage.conditioned medium Peritoneal macrophages were obtained from female rabbits (Nippon white) by intraperitoneal injection of 0.2% (w/v) glycogen, and the conditioned medium was prepared by costimulation of these cells with lipopolysaccharide as described previously [18]. Gel permeation chromatography indicated that there are three species with different molecular weights, all of which contain both the stimulatory effects on collagenase production and lymphocyte proliferation [9]. Cell culture and treatment Uterine cervices from women of child-bearing age were obtained from patients undergoing hysterectomy of fibromyoma not involving the cervix. The culture of stromal fibroblasts was established as described for cells from rabbit uterine cervix [19]. The cells were plated at a density of 1.10 s cells/ml of 10% (v/v) FCS/MEM in a 24 multiwell plate, and grown to confluence. In most experiments, cells with up to the fifth passage were used. After confluence, the collagenase production was ~nduced for 6 days by adding 50% (v/v) macrophageconditioned medium (MCM) or human recombinant IL-la (10 ng/ml) in the serum-free medium containing 0.2% (w/v) lactalbumin hydrolysate (LAH). Then the medium was replaced by W-7 and/or IL-l-supplemented medium and the culture was maintained for suitable periods.
Sti nulation of the IL-l-induced collagenase activity by co, :wd'.:!!,~ ,lntagonists Human uterine cervical fibroblasts in the culture produced no significant amount of collagenase, but the production of collagenase was stimulated by treating the cells with MCM or recombinant IL-la. Thus, to maintai,~ a constant level of collagenase production, .,'onfluent cells were first treated with 50% (v/v) MCM twice cor.secutively for 6 days, and they were then used for subsequent experiments. To e,~amine the effect of W-7 on MCM-induced collagenase production, fibroblasts primed with 50% (v/v) MCM were exposed to the reagent for 24 h in the presence of MCM. As shown in Fig. 1, the apparent collagenase activity detected in the culture media was stimulated by W-7 in a dose-dependent manner over the concentration range of 5-40 /~M. About 3-fold enhancement of collagenase production above the basal level was detected with 40 pM W-7. However, this enhancing effect of W-7 was not observed under the absence of IL-la (see below in Table I, Expt. 1) or MCM (data not shown). To confirm further the fact that the enhancing effect of W-7 on the MCM-induced procollagenase production is due to the inhibition of calmodulin, the effects of W-5 and TFP on the enzyme production were examined. W-5, a chloride-deficient analogue of W-7 that binds to calmodulin only weakly [17], showed no significant
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W-7orW-5 Concentration (~M) Fig. 1, The dose-dependent effect of W-7 or W-5 on the MCM-induced collagenase activity in human uterine cervical fibroblasts. Cervical fibroblasts at the third passage were maintained in 10% Iv/v) F C S / M E M until confluence, and then the culture medium was changed to 0.2% ( w / v ) L A H / M E M . The confluent cultures were first incubated with 50% (v/v) MCM as described in Materials and Methods. After 6 days, the cells were treated with W-7 or W-5 at the concentrations indicated (5-40 #M) for 24 h in the presence of 50,~ (v/v) MCM. The culture media were harvested for collagenase assay; o , W-7; ®o W-5. Each point represents the mean _+S.D. of three wells.
TABLE I
Effect of W-7 on the recombinant human IL-la- or MCM-dependent collagenase production by human uterine cervical fibroblasts Confluent cervical fibroblasts at primary (Expt. 1) or third passage (Expt. 2) were preincubated twice with 10 n g / m l recombinant human I L - l a or 50% ( v / v ) MCM for 6 days. Then the cells were treated with 40 laM W-7, 10 n g / m l I L - l a (or 50% (v/v) MCM) or W-7 plus IL-la (or MCM) in 0.2% ( w / v ) L A H / M E M . After 24 h, the culture media were harvested and assayed for collagenase activity, and then the cells were washed twice with phosphate-buffered saline and the cellular DNA content was determined as described in the text. Data are mean values_+ S.D. of three wells, n.d., not detectable. Treatment
Collagenase activity (units/ml)
Cellular DNA content (/t g/well)
Expt. 1 None W-7 (40/tM) It-1 a (10 n g / m l ) IL-la +W-7
n.d. n.d. 1.29 + 0.07 3.25+0.24 "
18.46 + 0.37 19.68 + 0.20 19.99 + 0.26 18.87+0.08
Expt. 2 None MCM (50% (v, v)) MCM +W-7 (40 ~M)
n.d. 2.24 __0.26 7.43_+1.29 b
16.25 + 0.15 17.80 _+0.24 17.55_+0.11
" Significant difference, P < 0.05, compared to corresponding value in IL-la. b Significant diff6;cnce, P < 0.Ol, compared to corresponding value in MCM.
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TFP Concentration (jiM) Fig. 2. Effect of T F P on MCM-induced collagenase production by Imman uterine cervical fibroblasts. Cervical fibroblasts at the third passage were incubated with 50f~ (v/v) MCM as described in Fig. !. Then the cells were exposed to various concentrations of TFP in the presence of 50e~ (v/v) MCM for 24 h. Each point repre.~cnts the mean + S.D. of three wclls.
stimulative effect on the coilagenase response to MCM (Fig. 1). In contrast, TFP, a calmodulin inhibitor of phenothiazine derivative [24], effectively promoted the MCM-induced collagenase activity to a similar extent as W-7 (Fig. 2). Although TFP has a relatively high hydrophobicity [14,15], and the nonspecific effects of TFP on cell membrane are not ruled out, the facts that W-7, a more specific plasma membrane-permeable calmodulin inhibitor, but not W-5 enhanced MCM-induced collagenase production and that TFP also stimulated the MCM-induced collagenase to a similar extent, suggest that calmodulin prevents the collagenase gene from being fully activated by MCM. A similar effect of W-7, but to a lesser extent, was also seen with cells treated with human recombinant IL-la (Table l), indicating that It-1 in MCM is a leading candidate ligand which induces collagenase. On the other hand, W-7 did not affect the cellular DNA content (Table I) and the cell number (data net show~). Therefore, the enhanced production of collagenase induced by W-7 was not due to the proliferation of ceils or the cytotoxic effect of the reagent. However, W-7 was not effective at concentrations higher than 50/.tM. At these concentrations, W-7 was found to be toxic to cervical fibroblasts: a considerable decrease in both cell number and cellular DNA content were observed (data not shown).
Effecl of W-7 on the production of procoilagenase It is well recognized that connective tissue cells in culture synthesize an endogenous metalloproteinase inhibitor (tissue inhibitor of metalloproteinases: TIMP) as
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Fig. 3. Enhanced production of procollagenase by W-7 from human uterine cervical fibroblasts. Confluent fibroblasts at the fourth passage were treated with 50% (v/v) MCM as described in Fig. 1. After 6 days, the cells were treated or not with 50% (v/v) MCM, or 40/,M W-7 plus MCM for 24 h. The harvested crude culture media (0.5 ml) were mixed with 0.25 mi of 10~ (w/v) trichloroacetic acid. The precipitates were collected by centrifugation, dissolved in 15 /,I of reducing sample buffer and subjected to SDS-PAGE. The procollagenase bands were visualized by immunoblotting as described in Materials and Methods, Lane !, no treatment: lane 2, MCM; lane 3, MCM plus W-7.
well as collagenase. Therefore, we examined that the enhanced collagenase activity found with W-7/MCM or IL-l-treated cells is due to the increased biosynthesis of the collagenase molecule. The amount of collagenase produ~;don wa~ measured by immunoblotting, using the specific antibody raised against human synovial collagnease. In the control culture, procollagenase was not detected immunologically (Fig. 3, lane 1) as predicted from the measurement of collagenase activity. When cervical fibroblasts were treated with MCM, procollagenase was obviously induced and detected as two species with Mr values of 55000 and 57000 (lane 2), and the production of procollagenase was augmented by the co-treatment of cells with W-7 (lane 3). This stimulative effect of W-7 on the procollagenase production was inhibited almost completely by 1 . 1 0 -5 M cycloheximide. Furthermore, W-7 did not cause any increase in the synthesis of the total cell proteins by human cervical fibroblasts (data not shown). Thus, the action of W-7 towards procollagenase production was
Fig. 4. Effect of W-7 and W-5 on the time course of the MCM-induced collagenase production by human uterine cervical fibroblasts. Cervical cells at the fifth passage were grown in 10% (v/v) FCS/MEM to confluence. Then the cells without MCM priming were treated with the following reagents in serum free MEM containing 0.2% (w/v) LAH and the media were changed daily for 6 days; o, W-7 (10/,M) and MCM (50% (v/v)); e, W-5 (10/,M) and MCM (50% (v/v)); A, MCM (50% (v/v)). Data are shown as the mean + S.D. of three wells.
concluded to be due to the direct modulation of the enzyme biosynthesis.
Effect of W-7 on the time course study of collagenase production T h e effect o f W - 7 o n I L - l - i n d u c e d c o ! ! 2 g e n a s e p r o d u c t i o n in cervical f i b r o b l a s t s was e x a m i n e d w i t h o u t
o I
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Days in culture Fig. 5. Influence of W-7 priming on the MCM-induced collagenase production in human uterine cervical fibroblasts. Confluent cervical cells at the second passage without MCM pretreatment were treated with W-7 in 0.2% (w/v) LAH/MEM for 24 h. Then the cells were maintained in 0.2% (w/v) LAH/MEM containing 50% (v/v) MCM, all the media were changed daily for 4 days. The abscissa indicates the days of culture after the removal of W-7. 4, 10 ~M W-7 primed; [2, 20 /tM, o, 40 ~M; A, non-primed control. Data are shown as the mean + S.D. of three wells.
65 MCM priming. Fig. 4 shows the time-dependent increase in collagenase production by 10 pM W-7 in the presence of 5070 (v/v) MCM. The same concentration of W-5, however, failed to enhance the collagenase production. These results indicate that the effect of W-7 in human cervical fibroblasts is also expressed by the consecutive treatment at a relatively low concentration of W-7 (10 #M), and is maintained when the cells coexists with W-7. On the other hand, in the absence of MCM, the cells produced no significant collagenase activity even after consecutive treatment with W-7 (10 #M) (data not shown). The responsiveness of the cervical cells pretreated with W-7 to MCM was also examined. The results are shown in Fig. 5. When the cells were primed with W-7 for 24 h, the inductive effect of MCM on collagenase production appeared more rapidly, in a dose-dependent manner, as compared with the cells with MCM alone. The enhancing effect of W-7 diminished 3 days after priming, indicating that this effect is reversible. Discussion
In the present study, we have demonstrated that the inhibition of calmodulin and the increase in the IL-l-induced collagenase production appear to be correlated. However, the exact roles of calmodulin on the increase in IL-l-induced collagenase are still unknown. We consider that there are at least three possibilities which could account for the above situation; calmodulin acts as (i) a mediator or regulator for initial signals of IL-1, (ii) a regulator for the collagenase gene expression and (iii) a regulator for the secretion of collagenase. If calmodulin is the intracellular mediator of IL-1, there might be a good correlation between IL-1 and the level of intracellular calcium after exposure to IL-I. We did not measure the cellular calcium concentration in human uterine cervical fibroblasts. However, Matsushima et al. [25] have reported an interesting observation that in murine thymocytes, calcium ionophore A23187 synergistically enhanced the thymocyte proliferation induced by IL-1, but A23187 alone caused no thymocyte proliferation. They have suggested in a recent review [1] that the expression of biological activity of IL-1 depends on the intracellular calcium concentration, but that it does not directly alter the intracellular level of calcium. Furthermore, Georgilis et al. [26] have reported that recombinant human IL-lfl had no effect on the concentration of intracellular free calcium in human neutrophils. In view of these observations and the fact that W-7 alone did not induce collagenase production in cervical cells (data not shown) suggest that it is unlikely that the effect of IL-1 on collagenase production in uterine cervical cells is due to the changes in the intracellular calcium level, and that calmodulin is an intracellular mediator of IL-1. On the other hand, IL-1
caused a slight transient fall in the total intracellular calcium level in a murine B-cell line [27]. Thus, the precise relationship between IL-1 and intracellul:~r calcium is not yet fully understood. It is also unlikely that calmodulin negatively regulates the secretion of collagenase from cervical cells, since the rate of the collagenase release into the extracellular space is reflected predominantly by its de novo synthesis [28,29]. Furthermore, our results show that pretreatment of the cells with W-7 can rapidly augment MCM-induced collagenase production (Fig. 5). Therefore, we censider the possibility that calmodulin may act by modulating collagenase gene expression rather than the secretion of collagenase. However, it is unclear how calmodulin regulates the production of collagenase. In this respect, the fact that caimodulin affects the cytoskeleton in various types ot cell [30] is of interest. In fact, Aggeler et al. [31] have reported that various agents which affect the cytoskeleton such as cytochalasin B, colchicin and TFP induced collagenase production in rabbit synovial fibroblasts by causing some changes in the cellular shape. These observations suggest that, in human uterine cervical cells, both TFP and W-7 may alter the interaction of ,he cytoskeleton with plasma membrane by binding to calmodulin, tbus resalting in the augmentation of the IL-l-induced collagenase production. However, these authors reported in their recent paper [32] that cycloheximide effectively inhibited the ocllagenase production, but it did not prevent these morphological changes induced by cytochalasin B. Furthermore, in human uterine cervical fibroblasts, the changes in cellular shape are not essential for the enhancement of IL-l-induced collagenase production rdata not shown). In conclusion, we have demonstrated that W-7, a specific calmodulin inhibitor, stimulates the IL-l-induced collagenase production in human uterine cervical fibroblasts. Although the precise mechanism is s61l unclear, we propose that calmodulin acts as a suppressor of collagenase production in these cells. Recently, a similar suppressive effect of calmodulin on the protein synthesis has also been reported for the synthesis of the low-density lipoprotein receptor in human skin fibroblasts [33]. Acknowledgment This study was supported in part by a National Institutes of Health Grant AR39189. Re|erences 10ppenheim. J.J., Kovacs, E.J., Matsushima. K. and Durum. S.K. (1986) lmmunol. Today 7, 45-56. 2 Dinarello.C.A. and Wolff, S.M. (li982) Am. J. Med. 72. 799-819. 3 Dinarello,C.A. (1984) Rev. Infect. Dis. 6, 51-95.
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