European Journal of Pharmacolo~,3' - Moh'cuNr Pha;'macology Section, 227 ( 19921 t 13-- 122
1! 3
~ 1992 Elsevier Science Publishers B.V. All rights reserved 0922-410c~/92/$05.00
EJPMOL 90348
Protein kinase C-dependent and -independent actions of a potent protein kinasc C inhibitor, staurosporine S~teshi Y a m a m o t o , H o n g Jiang, KJvotaka Nishikawa, Motoko Ishihara, Jian C h u n W a n g and Ryuichi Kato Department of Pharmaco:.ogy, School of Medicine, Keio Unirer~io', 35 Shimmomachi. Shinjuku-ku, Tokyo 160, Japan Received 4 December 1991, revised MS received 3 April 19~2, accepted 26 May 1992
12-O-Tetradccanoylphorbol 13-acetate (TPA), an activator of protein kinasc C (PKC), induced ornithinc decarboxylase (ODC) in primary cultured mouse epidermal cells. Staurosporinc, a potent protein kinasc C inhibitor, also induced ODC activity. Both TPA- and staurosporine-caused ODC inductions were markcdty suppressed in the PKC-down-regulatcd cells. Another PKC inhibitor, l-(5-isoquinolinesulfonyl)-2-methylpiperazinc (H-7), inl'dbimd both TPA- and staurosporinc-caused ODC inductions. H-7 by itself never induced ODC activity. Under our experimental conditions, staurosporine induced no detectable phosphorylation of endogenous proteins. TPA induced a iransloeation of PKC from cytosol ~ membrane whereas an optimal concentration of staurosporine to induce ODC did not induce an obvious translocation of PKC. Indomcthacin, a cyclooxygenase inhibitor, inhibited staurosporine-caused ODC induction, but not TPA-causcd ODC induction. Staurosporil~e induced specific morphological changes of epidermal cells both in normal and in PKC-down-regulated cells. These results indicate that staurosporinc induces ODC activity in a PKC-dependent manner and morphological changes possibly through a PKC-independent mechanism. The mechanism of ODC induction caused by staurosporine ma} be m some way different from that caused by TPA. Staurosporine; 12-O-Tctradccanoylphorbol 13-acetate; Protein kinase C; Ornithine decarboxylase: Epidermal cells (mouse); Morphological changes
I. Introduction
12-O-Tetradecanoylphorbol 13-acetate (TPA) is known as a very potent mouse skin tun, or promoter and induces a variety of biochemical changes in epidermal cells, the target ce!l~ of two-stage carcinogenesis in the skin. Protein kinase C (PKC) is regarded as a receptor for phorbol esters (Nishizuka, 1984), and an initial event irwolved in TPA action is believed to be the direct activation of this enzyme (Castagna ct aL, 1982; Nishizuka, 1984). Tamaoki et al. (1986) introduced a novel and very potent PKC inhibitor named staurosporine, a microbial alkaloid. Staurosporine potently inhibits mouse skin tumor promotion caused by TPA (Yamamoto et al., 1989). Since staurosporine actually inhibits PKC ob,tained either from cytosol or particulate fraction of mouse epidermis and also in-
Currespondence to: Dr. Satoshi Yamamoto, Department of Pharmacology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuk!,-t',!, "r'~kT~? l,q,q, .;apau. iei. 03-3353-1211 exl. 263(/: Fax 03-33598889.
hibits TPA-caused phosphory!ation of endogenous proteins in intact epidermal cells (Yamamoto et al,, 1980; Nishikawa et al., submitted for publication) and in vivo epidermis (Chida et al., 1988), it seems reasonable to speculate that the anti-tumor-promoting action of staurosporine is mediated through the inhibition of PKC by this agent. However, staurosporine unexpectedly fails to inhibit TPA-causeo epidermal hyperplasia, inflammation and ui-nithinc dccarboxylase (ODC) induction (Yamamoto et al., 1989), which are believed to be closely related to the process of skin tumor promotion (Boutweli, 1964; O'Brien, 1976; Scribner and Suss, 1978; Slaga et al., 1978; Sisskin et al., 1982; Kensler and Taffe, 19861. TPA-caused ODC induction in fleshly isolated mouse epidermal cells is also not inhibited by staurosporine (Kiyoto et al., 1987). Moreover, staurosporine by itself induces ODC activity in isolated epidermal cells (Kiyoto et al., 1987). In addition, it ha:" been reported that a very high dose of staurosporine by itsclf exerts a tumor-promoting effect (Yoshizawa et al., 199(1). The cause of such paradoxical cffccts of staurosporine has not been clarified yet.
114 In this study, v,e have investigated the mechanisms of ODC induction and mol~aological changes caused by staurosporine in primary cultured mouse cpidermal cells.
lured epidermal cells under low Ca 2+ (50/zM) conditions, in order to prevent the terminal differentiation of epidermal cells and to keep the cells as basal epidermal cells.
2.3. Ceil morphology 2. Materials and methods
2.1. Materials The following materials were purchased from the companies indicated: staurosporine, from Kyowa Medex Co., Tokyo, Japan; 1-(5-isoquinolinest,lfonyl)2-methylpiperazine (H-7), from Seikagaku Kogyo Co., Tokyo, Japan; Triton X-100, from Sigma Chemical Co., St. l,x)uis, MO, USA; DEAE-cellulose (DE52) from Whatman, Maidstone, UK; culture medium, from Gibco, Grand Island, NY, USA: fetal calf serum, from Hyclone, Logan, UT, USA; Bio-Lyte (5/7, 3/10) arnpho[ine, acrylamide and Che!ex 1()0 (200-400 mesh, soditim form), from Bio-Rad Lab., Richmond, CA, USA; TP~, from ~hemicals for Cancer Research, Chicago+ IL, USA; DL-[1-t4C]ornithine (58 m C i / retool), i~rom Amersham International, Buckinghamshire, UK; [~H]phorbol 12,13-dibutyrate (PDBu) (19 Ci/mmol), from New England Nuclear, Boston, MA, USA; [32p]H3PO 4, from Touho Biochemical Co., Tokyo, Japan. The other chemicals used were of reagent grade.
2.2. Prima~y culture of mouse epidermal cel& Primary cultured mouse epidermal cells were prepared according to the methods described by Yuspa (1985) with minor modifications (Nakadate et al., 1989; Yamamoto et al., 1989). Epidermal cells were isolated from new-born CD-I mice (1-2 days postpartum; Charles River, Atsugi, Japan) as described previously (Sasakawa et al., 1985). The cells were plated on plastic dishes at an initial density of 4 x 10 ~ cells/cm-" in Eagle's minimum essential medium sapp!emented with 10% fetal calf serum and were cultured at 36°C in at, atmosphere of 92% air/8% CO, for 20 h. Thereafter, the medium was switched to low Ca 2+ medium, i.e. Ca2~-free minimum essential medium supplemented with 10% chelex 100-treated (Ca- -deprived) fetal calf serum and 50 p.M CaCI 2 (final concentration), as described previously (Yuspa, 1985; Nakadate et al., 1989; Yamamoto et al., 1989). The culture medium contained the following antibiotics: 100 U / m l penicillin G, 100 # g / m l streptomycin sulfate and 0.25 # g / m l fungizone. The medium was changed daily and all experiments were performed with confluent cells at 4 days of culture. Cell viability was determined by the trypan blue exclusion test and was always above 90%. All the experiments have been done using these primary cul-
Morphol6gical changes of the cells were examined with a phase contrast microscope 3 h after the addition of drugs, unless otherwise indicated.
2.4. ODC induction On the day of experiments, the medium change was not performed, in order to prevent the ODC induction caused by the medium change, i.e. serum factor(s) (Nakadate et al., 1989). The primary cultured epidermal cells in 35-ram dishes were incubated for the indicated time periods at 36°C in an atmosphere of 92% a i r / 8 % CO 2 in the presence or absence of the indiea'~ed drugs. After the incubation, the cells were washed 3 times with Ca 2+- and Mg2+-free phosphate buffered saline (PBS(-)), frozen on dry ice and stored at - 8 0 ° C until assay. ODC assay was performed as described previously (Nakadate et al., 1989)~ The cells were sonicated in 50 mM sodium phosphate buffer, pH 7.2, containing 200 p.M pyridoxal 5'-phosphate and 50 /.tM EDTA, and centrifuged at 15,000 x g for 30 min. ODC activity of the supernatant was determined by measuring the release of 14CO2 from DL-[l-~4C]ornithine as described previously (Sasakawa et al., 1985; Nakadate et al., 1989).
2.5. Preparation of PKC-down-regulated cells PKC-down-regulated cells were prepared by treating the primary cultured epidermal cells with 100 nM TPA for 48 h in low Ca 2+ (50 ,aM) medium as described previously (Nakadate et al., 1989). The cells were then washed twice with P B S ( - ) and once with P B S ( - ) suppiemented with 0.i% bovine serum albumin ~ B" ~'~j '~ in order to wash out TFA. Using these PKC-down-regula~ed cells, the effects of staurosporine and TPA were also examined. All the experiments using the PKCdown-regulated cells were performed in a minimum essential medium (Ca 2 ' : 50/xM) containing 0.5% BSA but without fetal calf serum.
2.6. Translocation of PKC The primary cultured epidermal cells cultured in the low Ca 2+ (50 /xM) medium in 100-mm dishes were treated with the indicated agents for the indicated time periods. After the incubation, the amount of cellular PKC was determined by [3H]PDBu binding assay just as described previously (Nakadate et al., 1989). In
115
brief, the cells were senicated and centrifuged at 105,000 × g for 60 min. The resultant cytosol and particulate fractions were treated with Triton X-100. The Triton X-100-treated particulate fraction was centrifuged again at 105,000 × g for 60 rain. The resultant supernatant (solubilized particulate fraction) and Triton X-100-treated cytosol fraction were subjected to DEAE-cellulose (DE52) column chromatography. The amount of PKC eluted from the DE52 column was determined by [3H]PDBu binding assay (Nakadate et al., 1989), The binding reaction was carried out by incubating the sample solution with 15 nM [~H]PDBu in the presence of 2 mM Ca 2+ and 100 / z g / m l phosphatidylserine at 37°C for 5 min. Bound and free [3H]PDBu were separated by the polyethylene glycol precipitation method. For the estimation of non-specific binding, 30 ~ M unlabeled PDBu was added to the reaction mixture. Specific binding was calculated by subtracting the non-specific binding from the total binding.
2. 7. Protein phosphorylation in intact epidermal cells The primary cultured epidermal ce!!s 11.3 x l0 s c e l l s / m l / w e l l ) in 35-mm dishes were wasl~cd twice with phosphate-deprived Locke's solution (buffer A) of the following coraposition: 154 mM NaCI; 5.6 mM KCI; 0.05 mM CaCI2; 1 mM MgClz; 3.6 mM NaHCOs; 5.6 mM glucose; 10 mM HEPES (pH 7.4); 0.1% (w/v) BSA. Cells were first incubated in buffer A at 37°C for 30 rain. Then the medium was changed to 1 ml of new buffer A containing 0.2 m C i / m l [32p]H3PO 4, and the cells were further incubated for 30 min at 37°C. Thereafter the cells were washed once with buffer A and incubated in the same buffer with the indicated agents for 10 or 120 rain at 37°C. At the end of the incubation, the medium was removed by aspiration, and 0.25 ml sodium dodecyl sulfate (SDS) buffer (62.5 mM Tris, 2% SDS, 5% mercaptoethanol and 5% glycerol, pH 6.8) was added to the cells. Then the mixture was boiled for 5 rain.
2.8. Polyacrylamide gel electrophomsis (PAGE) 20 ttl aliquots of the boiled samples were subjected to SDSoPAGE (10% slab gel with 1.5-mm thickness). 200 ttl aliquots of boiled samples were analyzed by two-dimensional P A G E according to the method of O'Farrel[ 11975) with slight modifications. The first-dimensional isoelcctric focusing gels (5-mm diameter) contained 5% Bio-Lyte ampboiytes (pH 3-10) and the second-dimensional SDS-PAGE was performed using 10% polyacrylamide gels (l.5-mm thickness). These gels were fixed in a solution of 25% 1-propanol and 10% acetic acid for 1 h, stained with Coomassie blue and dried under vacunm. For autoradiography, dried
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Time (hr) Fig. 1. Time course of staurosporine-caused O D C induction in epidermal cells. Primary cul|ured mouse epidermal cells were: incubated with vehicle ( o ) or 30 nM ~taurosporine (o) for the indicated time periods in the low Ca 2 ~ (50 /.tM) medium and cellular O D C activity was determined. Each point and vertical bar represent mean _+S.E, (n ~ 4).
gels were cxposed to Kodak X A R film for 24-48 h at - 7 0 ° C with one intensifying screen.
2.9. Statistical analysis Statistical analysis was done by Student's t-test.
3. Results
Extracellular Ca -'+ is a regulator of the growth and differentiation of cultured mouse epidermal cells (Yuspa, 1985; Hennings et aI., 1980). Epidermal cells cultured in low Ca 2+ (51)/.tM) medium proliferated as basal cells. When medium Ca 2+ was raised to the normal level (1.8 raM), proliferation ceased and squamous differentiation ensued rapidly with sloughing of cells, as reported previously (Hennmgs et al., 1980: Yuspa, 1985). Therefore, all the experiments were performed under low C a 2+ (5(} t~M) conditions. Treatment of primary cultured epidermal cells with a potent PKC inhibitor, staurosporine, led to a transient induction of ODC activity, and a peak activity was observed 10 h after the addition of staurosporine to culture medium (fig. 1). The effect of staurosporine was concentration-dependent and a maximum response was obtained with 30 nM staurosporine (fig. 2). At a higher concentration, i.e. 60 nM, the activity induced was rather decreased (fig. 2). It has been established that the prolonged treatment of the cells with a potent PKC activator, TPA, markedly reduces the amount of PKC both in cytosol and particulate fractions, i.e. down-regulation of protein kinasc C (Nishizuka, 1988). In order to determine whether staurosporine exerts its effect in PKC down-regulated cells,
116 E 22 c
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Staurosporine (nM) Fig. 2. Concentration-response relationship of staurosporine-caused ODC induction in epidermal cells. Primary cultured mouse epidermal ceils were incubated with the indicated concenua!ions of staurosporine for 10 h in the low Ca 2~ (50 /tM) medium and celhdar ODC activity was determined. Each point and vertical bar represent mean ±S.E. (n = 4). * P < ().Ill versus 1).
the following e x p e r i m e n t s were perfi3rmcd. T h e cells were first t r e a t e d with 100 n M T P A for 48 h in o r d e r to d o w n - r e g u l a t e PKC, t h e n the a m o u n t of cellular P K C was e s t i m a t e d by [ 3 H ] P D B u b i n d i n g to this enzyme. A l t h o u g h c o m p l e t e d o w n - r e g u l a t i o n was not observed, the specific [ 3 H ] P D B u binding e i t h e r in cytosol or particulate fraction was markedly r e d u c e d (approximatei:¢ 20% or less of control). W e have previously r e p o r t e d that T P A induces O D C activity in the primary c u l t u r e d m o u s e e p i d e r m a l cells and the p e a k activity is o b s e r v e d at 3 h after T P A t r e a t m e n t (Nakadate et al., 1989). T h e maximal activity is i n d u c e d by 30 n M T P A ( N a k a d a t e et al., 1989). T h e r e f o r e , O D C activity was d e t e r m i n e d 3 h a f t e r t h e addition of 30 n M T P A into culture m e d i u m . As shown i~ fig. 3, T P A i n d u c e d m u c h h i g h e r O D C activity t h a n s t a u r o s p o r i n e in the control cells (without PKC down-regulation). A s can be expected, O D C induction caused by T P A disapp e a r e d completely in the P K C - d o w n - r e g u l a t e d cells (fig. 3). In the same P K C - d o w n - r e g u l a t e d cells, staur o s p o r i n e - c a u s e d O D C induction was also m a r k e d l y s u p p r e s s e d (fig. 4). T h e effect of H-7, a well-known PKC inhibitor ( H i d a k a et al., 1984), on s t a u r o s p o r i n e - c a u s e d O D C induction was investigated in t h e n o r ~ a I primary cult u r e d e p i d e r m a l cells. H-7 i n h i b i t e d b o t h T P A - a n d s t a u r o s p o r i n e - c a u s e d O D C induction in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r at a similar c o n c e n t r a t i o n r a n g e (fig. 5). H-7 did not shift the time cours~ of O D C induction caused by s t a u r o s p o r i n e ( d a t a not shown). H-7 by itself never induced O D C activity ( d a t a not shown).
0 PKC down-regulation TPA stimulation
--
+
+
+
+
-
Fig. 3. Failure of TPA to induce ODC activity in PKC-down-regulated epidermal cells. Primary cultured mouse epidermal cells weJe preincubated with vehicle or 100 nM TPA for 48 h in the low Ca 2+ (50 #M) medium. Thereafter, the cells were washed and the medium was replaced with a new low Ca 2 + (50/zM) medium containing 0.5% BSA but without fetal calf serum. The cells were further incubated for 3 h with vehicle or 30 nM TPA and cellular ODC activity was determined. Each column and vertical bar represent mean±S.E. (n = 4). * ? < (H)! versus PKC d~.~wn-regulation ( - ), TPA stimulalion ( + ). and no! significant!y different from TPA stimulation ( - ).
T h e effects of i n d o m e t h a c i n , a cyclooxygenase inhibitor, o n T P A - a n d s t a u r o s p o r i n e - c a u s e d O D C induction were also examined. A s s h o w n in fig. 6, 1 t i M i n d o m e t h a c i n i n h i b i t e d staur~spc:rine-caused O D C induction. T h i s c o n c e n t r a t i o n ot i n d o m e t h a c i n completely s u p p r e s s e s P G E 2 synthesis in intact e p i d e r m a l cells (Aizu et al., 1990). O n the o t h e r h a n d , T P A - c a u s e d O D C induction was not i n h i b i t e d by i n d o m e t h a c i n . T h e result is c o n s i s t e n t with o u r previous r e p o r t which 0.4 A
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+ + PKC down-regulation t + Staurosporine stimulation Fig. 4. Failure of staurosporine to induce ODC activity in PKCdown-regulated epidermal cells. Primary cultured mouse epidermal cells were preincubated with vehicle or 100 nM TPA for 48 h in the low Ca 2~ (50 ~M) medium. Thereafter, the cells were washed and the medium was replaced with a new low Ca ~ (50 i.tM) medium containing 0.5% BSA but without fetal calf serum. The cells were further incubated liar 10 h with vehicle or 30 nM staurosporine and celhdar ODC activity was determined. Each column and vertical bar represent mean +_.S.E. (n=4). * P
1! 3
~ 1992 Elsevier Science Publishers B.V. All rights reserved 0922-410c~/92/$05.00
EJPMOL 90348
Protein kinase C-dependent and -independent actions of a potent protein kinasc C inhibitor, staurosporine S~teshi Y a m a m o t o , H o n g Jiang, KJvotaka Nishikawa, Motoko Ishihara, Jian C h u n W a n g and Ryuichi Kato Department of Pharmaco:.ogy, School of Medicine, Keio Unirer~io', 35 Shimmomachi. Shinjuku-ku, Tokyo 160, Japan Received 4 December 1991, revised MS received 3 April 19~2, accepted 26 May 1992
12-O-Tetradccanoylphorbol 13-acetate (TPA), an activator of protein kinasc C (PKC), induced ornithinc decarboxylase (ODC) in primary cultured mouse epidermal cells. Staurosporinc, a potent protein kinasc C inhibitor, also induced ODC activity. Both TPA- and staurosporine-caused ODC inductions were markcdty suppressed in the PKC-down-regulatcd cells. Another PKC inhibitor, l-(5-isoquinolinesulfonyl)-2-methylpiperazinc (H-7), inl'dbimd both TPA- and staurosporinc-caused ODC inductions. H-7 by itself never induced ODC activity. Under our experimental conditions, staurosporine induced no detectable phosphorylation of endogenous proteins. TPA induced a iransloeation of PKC from cytosol ~ membrane whereas an optimal concentration of staurosporine to induce ODC did not induce an obvious translocation of PKC. Indomcthacin, a cyclooxygenase inhibitor, inhibited staurosporine-caused ODC induction, but not TPA-causcd ODC induction. Staurosporil~e induced specific morphological changes of epidermal cells both in normal and in PKC-down-regulated cells. These results indicate that staurosporinc induces ODC activity in a PKC-dependent manner and morphological changes possibly through a PKC-independent mechanism. The mechanism of ODC induction caused by staurosporine ma} be m some way different from that caused by TPA. Staurosporine; 12-O-Tctradccanoylphorbol 13-acetate; Protein kinase C; Ornithine decarboxylase: Epidermal cells (mouse); Morphological changes
I. Introduction
12-O-Tetradecanoylphorbol 13-acetate (TPA) is known as a very potent mouse skin tun, or promoter and induces a variety of biochemical changes in epidermal cells, the target ce!l~ of two-stage carcinogenesis in the skin. Protein kinase C (PKC) is regarded as a receptor for phorbol esters (Nishizuka, 1984), and an initial event irwolved in TPA action is believed to be the direct activation of this enzyme (Castagna ct aL, 1982; Nishizuka, 1984). Tamaoki et al. (1986) introduced a novel and very potent PKC inhibitor named staurosporine, a microbial alkaloid. Staurosporine potently inhibits mouse skin tumor promotion caused by TPA (Yamamoto et al., 1989). Since staurosporine actually inhibits PKC ob,tained either from cytosol or particulate fraction of mouse epidermis and also in-
Currespondence to: Dr. Satoshi Yamamoto, Department of Pharmacology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuk!,-t',!, "r'~kT~? l,q,q, .;apau. iei. 03-3353-1211 exl. 263(/: Fax 03-33598889.
hibits TPA-caused phosphory!ation of endogenous proteins in intact epidermal cells (Yamamoto et al,, 1980; Nishikawa et al., submitted for publication) and in vivo epidermis (Chida et al., 1988), it seems reasonable to speculate that the anti-tumor-promoting action of staurosporine is mediated through the inhibition of PKC by this agent. However, staurosporine unexpectedly fails to inhibit TPA-causeo epidermal hyperplasia, inflammation and ui-nithinc dccarboxylase (ODC) induction (Yamamoto et al., 1989), which are believed to be closely related to the process of skin tumor promotion (Boutweli, 1964; O'Brien, 1976; Scribner and Suss, 1978; Slaga et al., 1978; Sisskin et al., 1982; Kensler and Taffe, 19861. TPA-caused ODC induction in fleshly isolated mouse epidermal cells is also not inhibited by staurosporine (Kiyoto et al., 1987). Moreover, staurosporine by itself induces ODC activity in isolated epidermal cells (Kiyoto et al., 1987). In addition, it ha:" been reported that a very high dose of staurosporine by itsclf exerts a tumor-promoting effect (Yoshizawa et al., 199(1). The cause of such paradoxical cffccts of staurosporine has not been clarified yet.
114 In this study, v,e have investigated the mechanisms of ODC induction and mol~aological changes caused by staurosporine in primary cultured mouse cpidermal cells.
lured epidermal cells under low Ca 2+ (50/zM) conditions, in order to prevent the terminal differentiation of epidermal cells and to keep the cells as basal epidermal cells.
2.3. Ceil morphology 2. Materials and methods
2.1. Materials The following materials were purchased from the companies indicated: staurosporine, from Kyowa Medex Co., Tokyo, Japan; 1-(5-isoquinolinest,lfonyl)2-methylpiperazine (H-7), from Seikagaku Kogyo Co., Tokyo, Japan; Triton X-100, from Sigma Chemical Co., St. l,x)uis, MO, USA; DEAE-cellulose (DE52) from Whatman, Maidstone, UK; culture medium, from Gibco, Grand Island, NY, USA: fetal calf serum, from Hyclone, Logan, UT, USA; Bio-Lyte (5/7, 3/10) arnpho[ine, acrylamide and Che!ex 1()0 (200-400 mesh, soditim form), from Bio-Rad Lab., Richmond, CA, USA; TP~, from ~hemicals for Cancer Research, Chicago+ IL, USA; DL-[1-t4C]ornithine (58 m C i / retool), i~rom Amersham International, Buckinghamshire, UK; [~H]phorbol 12,13-dibutyrate (PDBu) (19 Ci/mmol), from New England Nuclear, Boston, MA, USA; [32p]H3PO 4, from Touho Biochemical Co., Tokyo, Japan. The other chemicals used were of reagent grade.
2.2. Prima~y culture of mouse epidermal cel& Primary cultured mouse epidermal cells were prepared according to the methods described by Yuspa (1985) with minor modifications (Nakadate et al., 1989; Yamamoto et al., 1989). Epidermal cells were isolated from new-born CD-I mice (1-2 days postpartum; Charles River, Atsugi, Japan) as described previously (Sasakawa et al., 1985). The cells were plated on plastic dishes at an initial density of 4 x 10 ~ cells/cm-" in Eagle's minimum essential medium sapp!emented with 10% fetal calf serum and were cultured at 36°C in at, atmosphere of 92% air/8% CO, for 20 h. Thereafter, the medium was switched to low Ca 2+ medium, i.e. Ca2~-free minimum essential medium supplemented with 10% chelex 100-treated (Ca- -deprived) fetal calf serum and 50 p.M CaCI 2 (final concentration), as described previously (Yuspa, 1985; Nakadate et al., 1989; Yamamoto et al., 1989). The culture medium contained the following antibiotics: 100 U / m l penicillin G, 100 # g / m l streptomycin sulfate and 0.25 # g / m l fungizone. The medium was changed daily and all experiments were performed with confluent cells at 4 days of culture. Cell viability was determined by the trypan blue exclusion test and was always above 90%. All the experiments have been done using these primary cul-
Morphol6gical changes of the cells were examined with a phase contrast microscope 3 h after the addition of drugs, unless otherwise indicated.
2.4. ODC induction On the day of experiments, the medium change was not performed, in order to prevent the ODC induction caused by the medium change, i.e. serum factor(s) (Nakadate et al., 1989). The primary cultured epidermal cells in 35-ram dishes were incubated for the indicated time periods at 36°C in an atmosphere of 92% a i r / 8 % CO 2 in the presence or absence of the indiea'~ed drugs. After the incubation, the cells were washed 3 times with Ca 2+- and Mg2+-free phosphate buffered saline (PBS(-)), frozen on dry ice and stored at - 8 0 ° C until assay. ODC assay was performed as described previously (Nakadate et al., 1989)~ The cells were sonicated in 50 mM sodium phosphate buffer, pH 7.2, containing 200 p.M pyridoxal 5'-phosphate and 50 /.tM EDTA, and centrifuged at 15,000 x g for 30 min. ODC activity of the supernatant was determined by measuring the release of 14CO2 from DL-[l-~4C]ornithine as described previously (Sasakawa et al., 1985; Nakadate et al., 1989).
2.5. Preparation of PKC-down-regulated cells PKC-down-regulated cells were prepared by treating the primary cultured epidermal cells with 100 nM TPA for 48 h in low Ca 2+ (50 ,aM) medium as described previously (Nakadate et al., 1989). The cells were then washed twice with P B S ( - ) and once with P B S ( - ) suppiemented with 0.i% bovine serum albumin ~ B" ~'~j '~ in order to wash out TFA. Using these PKC-down-regula~ed cells, the effects of staurosporine and TPA were also examined. All the experiments using the PKCdown-regulated cells were performed in a minimum essential medium (Ca 2 ' : 50/xM) containing 0.5% BSA but without fetal calf serum.
2.6. Translocation of PKC The primary cultured epidermal cells cultured in the low Ca 2+ (50 /xM) medium in 100-mm dishes were treated with the indicated agents for the indicated time periods. After the incubation, the amount of cellular PKC was determined by [3H]PDBu binding assay just as described previously (Nakadate et al., 1989). In
115
brief, the cells were senicated and centrifuged at 105,000 × g for 60 min. The resultant cytosol and particulate fractions were treated with Triton X-100. The Triton X-100-treated particulate fraction was centrifuged again at 105,000 × g for 60 rain. The resultant supernatant (solubilized particulate fraction) and Triton X-100-treated cytosol fraction were subjected to DEAE-cellulose (DE52) column chromatography. The amount of PKC eluted from the DE52 column was determined by [3H]PDBu binding assay (Nakadate et al., 1989), The binding reaction was carried out by incubating the sample solution with 15 nM [~H]PDBu in the presence of 2 mM Ca 2+ and 100 / z g / m l phosphatidylserine at 37°C for 5 min. Bound and free [3H]PDBu were separated by the polyethylene glycol precipitation method. For the estimation of non-specific binding, 30 ~ M unlabeled PDBu was added to the reaction mixture. Specific binding was calculated by subtracting the non-specific binding from the total binding.
2. 7. Protein phosphorylation in intact epidermal cells The primary cultured epidermal ce!!s 11.3 x l0 s c e l l s / m l / w e l l ) in 35-mm dishes were wasl~cd twice with phosphate-deprived Locke's solution (buffer A) of the following coraposition: 154 mM NaCI; 5.6 mM KCI; 0.05 mM CaCI2; 1 mM MgClz; 3.6 mM NaHCOs; 5.6 mM glucose; 10 mM HEPES (pH 7.4); 0.1% (w/v) BSA. Cells were first incubated in buffer A at 37°C for 30 rain. Then the medium was changed to 1 ml of new buffer A containing 0.2 m C i / m l [32p]H3PO 4, and the cells were further incubated for 30 min at 37°C. Thereafter the cells were washed once with buffer A and incubated in the same buffer with the indicated agents for 10 or 120 rain at 37°C. At the end of the incubation, the medium was removed by aspiration, and 0.25 ml sodium dodecyl sulfate (SDS) buffer (62.5 mM Tris, 2% SDS, 5% mercaptoethanol and 5% glycerol, pH 6.8) was added to the cells. Then the mixture was boiled for 5 rain.
2.8. Polyacrylamide gel electrophomsis (PAGE) 20 ttl aliquots of the boiled samples were subjected to SDSoPAGE (10% slab gel with 1.5-mm thickness). 200 ttl aliquots of boiled samples were analyzed by two-dimensional P A G E according to the method of O'Farrel[ 11975) with slight modifications. The first-dimensional isoelcctric focusing gels (5-mm diameter) contained 5% Bio-Lyte ampboiytes (pH 3-10) and the second-dimensional SDS-PAGE was performed using 10% polyacrylamide gels (l.5-mm thickness). These gels were fixed in a solution of 25% 1-propanol and 10% acetic acid for 1 h, stained with Coomassie blue and dried under vacunm. For autoradiography, dried
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Time (hr) Fig. 1. Time course of staurosporine-caused O D C induction in epidermal cells. Primary cul|ured mouse epidermal cells were: incubated with vehicle ( o ) or 30 nM ~taurosporine (o) for the indicated time periods in the low Ca 2 ~ (50 /.tM) medium and cellular O D C activity was determined. Each point and vertical bar represent mean _+S.E, (n ~ 4).
gels were cxposed to Kodak X A R film for 24-48 h at - 7 0 ° C with one intensifying screen.
2.9. Statistical analysis Statistical analysis was done by Student's t-test.
3. Results
Extracellular Ca -'+ is a regulator of the growth and differentiation of cultured mouse epidermal cells (Yuspa, 1985; Hennings et aI., 1980). Epidermal cells cultured in low Ca 2+ (51)/.tM) medium proliferated as basal cells. When medium Ca 2+ was raised to the normal level (1.8 raM), proliferation ceased and squamous differentiation ensued rapidly with sloughing of cells, as reported previously (Hennmgs et al., 1980: Yuspa, 1985). Therefore, all the experiments were performed under low C a 2+ (5(} t~M) conditions. Treatment of primary cultured epidermal cells with a potent PKC inhibitor, staurosporine, led to a transient induction of ODC activity, and a peak activity was observed 10 h after the addition of staurosporine to culture medium (fig. 1). The effect of staurosporine was concentration-dependent and a maximum response was obtained with 30 nM staurosporine (fig. 2). At a higher concentration, i.e. 60 nM, the activity induced was rather decreased (fig. 2). It has been established that the prolonged treatment of the cells with a potent PKC activator, TPA, markedly reduces the amount of PKC both in cytosol and particulate fractions, i.e. down-regulation of protein kinasc C (Nishizuka, 1988). In order to determine whether staurosporine exerts its effect in PKC down-regulated cells,
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Staurosporine (nM) Fig. 2. Concentration-response relationship of staurosporine-caused ODC induction in epidermal cells. Primary cultured mouse epidermal ceils were incubated with the indicated concenua!ions of staurosporine for 10 h in the low Ca 2~ (50 /tM) medium and celhdar ODC activity was determined. Each point and vertical bar represent mean ±S.E. (n = 4). * P < ().Ill versus 1).
the following e x p e r i m e n t s were perfi3rmcd. T h e cells were first t r e a t e d with 100 n M T P A for 48 h in o r d e r to d o w n - r e g u l a t e PKC, t h e n the a m o u n t of cellular P K C was e s t i m a t e d by [ 3 H ] P D B u b i n d i n g to this enzyme. A l t h o u g h c o m p l e t e d o w n - r e g u l a t i o n was not observed, the specific [ 3 H ] P D B u binding e i t h e r in cytosol or particulate fraction was markedly r e d u c e d (approximatei:¢ 20% or less of control). W e have previously r e p o r t e d that T P A induces O D C activity in the primary c u l t u r e d m o u s e e p i d e r m a l cells and the p e a k activity is o b s e r v e d at 3 h after T P A t r e a t m e n t (Nakadate et al., 1989). T h e maximal activity is i n d u c e d by 30 n M T P A ( N a k a d a t e et al., 1989). T h e r e f o r e , O D C activity was d e t e r m i n e d 3 h a f t e r t h e addition of 30 n M T P A into culture m e d i u m . As shown i~ fig. 3, T P A i n d u c e d m u c h h i g h e r O D C activity t h a n s t a u r o s p o r i n e in the control cells (without PKC down-regulation). A s can be expected, O D C induction caused by T P A disapp e a r e d completely in the P K C - d o w n - r e g u l a t e d cells (fig. 3). In the same P K C - d o w n - r e g u l a t e d cells, staur o s p o r i n e - c a u s e d O D C induction was also m a r k e d l y s u p p r e s s e d (fig. 4). T h e effect of H-7, a well-known PKC inhibitor ( H i d a k a et al., 1984), on s t a u r o s p o r i n e - c a u s e d O D C induction was investigated in t h e n o r ~ a I primary cult u r e d e p i d e r m a l cells. H-7 i n h i b i t e d b o t h T P A - a n d s t a u r o s p o r i n e - c a u s e d O D C induction in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r at a similar c o n c e n t r a t i o n r a n g e (fig. 5). H-7 did not shift the time cours~ of O D C induction caused by s t a u r o s p o r i n e ( d a t a not shown). H-7 by itself never induced O D C activity ( d a t a not shown).
0 PKC down-regulation TPA stimulation
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Fig. 3. Failure of TPA to induce ODC activity in PKC-down-regulated epidermal cells. Primary cultured mouse epidermal cells weJe preincubated with vehicle or 100 nM TPA for 48 h in the low Ca 2+ (50 #M) medium. Thereafter, the cells were washed and the medium was replaced with a new low Ca 2 + (50/zM) medium containing 0.5% BSA but without fetal calf serum. The cells were further incubated for 3 h with vehicle or 30 nM TPA and cellular ODC activity was determined. Each column and vertical bar represent mean±S.E. (n = 4). * ? < (H)! versus PKC d~.~wn-regulation ( - ), TPA stimulalion ( + ). and no! significant!y different from TPA stimulation ( - ).
T h e effects of i n d o m e t h a c i n , a cyclooxygenase inhibitor, o n T P A - a n d s t a u r o s p o r i n e - c a u s e d O D C induction were also examined. A s s h o w n in fig. 6, 1 t i M i n d o m e t h a c i n i n h i b i t e d staur~spc:rine-caused O D C induction. T h i s c o n c e n t r a t i o n ot i n d o m e t h a c i n completely s u p p r e s s e s P G E 2 synthesis in intact e p i d e r m a l cells (Aizu et al., 1990). O n the o t h e r h a n d , T P A - c a u s e d O D C induction was not i n h i b i t e d by i n d o m e t h a c i n . T h e result is c o n s i s t e n t with o u r previous r e p o r t which 0.4 A
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+ + PKC down-regulation t + Staurosporine stimulation Fig. 4. Failure of staurosporine to induce ODC activity in PKCdown-regulated epidermal cells. Primary cultured mouse epidermal cells were preincubated with vehicle or 100 nM TPA for 48 h in the low Ca 2~ (50 ~M) medium. Thereafter, the cells were washed and the medium was replaced with a new low Ca ~ (50 i.tM) medium containing 0.5% BSA but without fetal calf serum. The cells were further incubated liar 10 h with vehicle or 30 nM staurosporine and celhdar ODC activity was determined. Each column and vertical bar represent mean +_.S.E. (n=4). * P
