Involvement of protein kinase C in bradykinin-induced intracellular calcium increase in primary cultured human keratinocytes

JOURNALOF

Dermatological Science Journal of Dematological Science 9 (1995) 11l-l 16

ELSEVIER

Involvement of protein kinase C in bradykinin-induced intracellular calcium increase in primary cultured human keratinocytes Y. Aoyamaa, M. Seishima*a, S. Maria, Y. Kitajima”, Y. Okanob, Y. NozawaC aDepartment of Dermatology, Gifu University School of Medicine Tsukasamachi 40, G~jii City 500, Japan ‘Department of Molecular Pathobiochemistry, Gtjii University School of Medicine, Gifu, Japan ‘Department of Biochemistry, GI~U University School of Medicine, Gifu, Japan

Received14January 1994;revision received11August 1994;accepted30 August 1994

Abstract Bradykinin (BK) is one of the key mediators of inflammation and a weak mitogen. We have previously demonstrated that BK induced the generation of inositol 1,4,5trisphosphate (Ins(l,4,5)P,) which caused Ca” mobilization in human keratinocytes. In this study, BK-induced Ca*+ responseswere examined in primary cultured human keratinocytes by video imaging fluorescence microscopy using fura-2. Intracellular calcium concentration ([Ca2+li) level increasedto a peak within 30 s after BK addition and decreasedgradually to the basal level. The existenceof the broad shoulder in the [Ca*+& profile was suggestedto be due to the Ca’+ influx from the external medium, becausethis disappeared in the presenceof 0.5 mM EGTA. Pretreatment with phorbol-lZmyristate-13-acetate (PMA), a protein kinase C (PKC) activator, significantly resulted in reduction of the descending shoulder of BK-induced increase in [Ca*‘]i. A 20-min pretreatment with PKC inhibitors, H-7 or staurosporine, reversed the decreaseby PMA in the shoulder of BK-induced Ca*+ response. Furthermore, the BK-induced [4’Ca] uptake was inhibited by EGTA and PMA. Ins(1,4,5)P, generation induced by BK peaked at 20 s and returned to the basal level at 60 s. There were no significant differences in Ins(1,4,5)P, levels at 20 and 60 s among the cells exposed to BK alone, BK with PMA pretreatment (20 min) and BK with PMA + H-7 pretreatment. These results suggestthat the BK-induced Ca*+ influx, which was shown as shoulder, may be negatively modulated by PKC in primary cultured human keratinocytes. Keywords:

Intracellular calcium; Human keratinocyte; Bradykinin

1. Introduction

many tissues or cell types [l]. Rosenbach and

The nanopeptide bradykinin (BK) is one of the key mediators of inflammation and is a mitogen. The receptors for BK are widely distributed in

Greenblee [2] observed that BK stimulated the generation of inositol 1,4,%trisphosphate (Ins (1,4,5)P,) in a human squamous cell carcinoma cell line (SCC-12F) and an immortalized cell line (HaCaT) keratinocytes [3]. Binding of growth factors or mitogens to their receptors ac-

* Correspondingauthor.

0923-181l/95/$09:50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0923-1811(94)00359-M

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tivates various signal transduction pathways, including the phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol bisphosphate (PIP,) to produce two second messengers, Ins( 1,4,5)P3 and 1,2-diacylglycerol (DAG) [4]. DAG remains in the plasma membrane and acts as an endogenous activator of protein kinase C (PKC), which in turn phosphorylates specific proteins, whereas Ins(l,4,5)Ps is releasedinto cytosol and mobilizes Ca*+ from intracellular storage sites. The Ins(l,4,5)P3-mediated Ca*+ signal results in activation of calmodulin-dependent protein kinase and several other Ca*+-dependent protein kinases and enzymes [5,6]. These signaling events are thought to be involved in cell proliferation and differentiation. Increases in intracellular Ca*+ play an important role at the early event of terminal differentiation in keratinocytes [4,7]. Although substantial evidencefor the phosphoinositide-mediated signal transduction pathway has been available for a wide variety of cells, there are relatively few reports dealing with this signal transduction in human keratinocytes. In this study, we have examined BK-induced calcium response, Ins( 1,4,5)P, generation and [45Ca] uptake in primary cultured human keratinocytes, and suggested that PKC may negatively modulate Ca*+ responses. 2. Materials and methods 2.1. Materials

Phorbol-l2-myristate-13-acetate (PMA), and bradykinin (BK) were obtained from Sigma Chemical Co. (St. Louis, MO). The acetoxymethyl ester of fura- (fura-2/AM) was obtained from Dojin Laboratories (Kumamoto, Japan). Staurosporine and 1-(5-isoquinoline sulphonyl)-2methylpiperazine dihydrochloride (H-7) were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Bovine pituitary extract (BPE) containing KGM serum-free medium (Kurrabou Co. Osaka, Japan) were used for cell culture. The quantitative assay kit for Dmyoinositol 1,4,5-trisphosphate (Ins(l,4,5)P3) was obtained from Amersham. All other chemicals were of reagent grade.

2.2. Cell culture Skin sampleswere obtained from breast skin of 14 normal healthy volunteers without any skin diseases (sex: two males and 12 females, age: 52.3 f 9.5, n = 14). The skin was trimmed of fat and incubated at 4°C in Hank’s balanced salt solution (HBSS) containing 500 U/ml dispase for 12 h to separate epidermis from dermis. Then the epidermis was incubated in HBSS with 0.002% EDTA at 37°C for 20 min. After washing with HBSS, the cells were plated at an initial density of 1 x 10’ cells/well of the Flexiparm-Disc (Heraeus Biotechnologie, Germany) in BPE containing KGM serum-free medium (0.15 mM Ca*‘), and cultured for 3 days at 37°C in humidified 5% C02/95% air. The cells were then washed once with HBSS, and treated with the BPE-free KGM medium for 24 h. All experiments were done with subconfluent cultured cells 4-5 days after plating. 2.3. Measurement of [ Ca2+li in single cells

The cells prepared as above were washed with Hepes buffer (25 mM Na-Hepes pH 7.4, 120 mM NaCl, 5 mM KCI, 1 mM MgCl,, 10 mM glucose) and were loaded with a fluorescence indicator fura-UAM (5 PM) for 2 h at 25°C in 0.2 ml of HEPES buffer. They were rinsed with HEPES buffer to remove free extracellular dye and were incubated for 15 min to allow deesteritication of the dye. Fluorescence intensity was measured for single cells at 37°C (3-6 s per reading) with excitation wavelengths of 340 and 360 nm on a fluorescence image analyser ARGUS- 1OO/CA (Hamamatsu Photonics Corp., Hamamatsu, Japan). The ratio of fluorescence intensity 3401360 nm was converted to [Ca*+]i by a calculation using a standard curve prepared in Ca*+-EGTA buffer as previously described [8]. 2.4. Ins(1,4,5) P3 measurement

The assayfor Ins( 1,4,5)P3was performed using the Ins(l,4,5)Ps assay kit as described previously [9]. For the quantitation of Ins(l,4,5)P3 levels, human keratinocytes in 24-well culture trays were preincubated at 37°C in 500 ~1 of low-calcium (25 PM) Eagle’s MEM containing 10 mM LiCl for 10 min, and then the medium was quickly aspirated

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and bradykinin in L-MEM was added. The reaction was terminated at different intervals by adding ice-cold 10%perchloric acid and the reaction mixture was then neutralized by the addition of 1.53 M KOH containing 75 mM HEPES. The solution was kept on ice for 1 h, transferred from the tray to microtubes and centrifuged at 2000 x g for 15 min at 4°C. The supernatant was further centrifuged at 12 000 x g for 15min at 4°C. The final supematant was added to the Ins( 1,4,5)Ps assay mixture containing a specific Ins( 1,4,5)P3-binding protein (Amersham, UK) and was incubated for 15 min at 4°C prior to centrifugation at 2000 X g for 15 min. The resulting pellets were resuspended in water and aliquots were measured for the radioactivity. The amount of Ins(1,4,5)P, was determined from the standard curve of authentic Ins( 1,4,5)Ps. 2.5. Measurement of 4sCa2+ uptake

Cultured keratinocytes were detached from the culture dishes using 0.05% (w/v) trypsin-0.02 mM EDTA. Following inactivation of trypsin by the addition of 0.1% (w/v) soybean trypsin inhibitor (Sigma, St. Louis, MO), the cells were collected by low-speed centrifugation and resuspended in defined medium to a final cell-density of 1.5 x lo6 cells/ml. After equilibration in a waterbath to 37°C test substances were, added with tracer amounts of 45CaC12(Amersham Corp. Arlington Heights, IL). The uptake of tracer was terminated by the addition of an excessof ice-cold stop buffer which contained 144 mM NaCl, 5 mM CaCl,, 5 mM Tris-HCl (pH 7.4) and the immediate filtration of the cell suspension through Whatman GF/C glass fibre filters, which were rapidly rinsed three times with 5 ml of stop buffer. The filters were air-dried, and the amount of cell-associated radioactivity was determined in a Beckman LS7500 scintillation spectrometer. 2.6. Statistical analysis

Statistical analysis of the data was carried out by Students’ unpaired tr:est. In the measurement’ of [Ca2+]i, eight cells in each case were used for measurement and their averageswere calculated.

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3. Results and discussion More than 80% of the cells showed [Ca2+]i increase to 200 nM BK. A representative trace of [Ca2+]i is shown in Fig. la. The [Ca2+]i level abruptly increased to a peak (354 f 120 nM, mean f S.D., n = 7 cases)within 30 s after BK stimulation and decreasedgradually to the basal level over 3 min. In the presence of extracellular EGTA (0.5 mM), although the first peak of [Ca2+]iwas not significantly changed, the slow descending phasewas markedly reduced to the basal level within 60 s (98 f 28 nM) (P < 0.001) (Fig. lb). Significant difference was thus observed in the BK-induced [Ca2+]i profile in the absence and in the presenceof EGTA (Fig. 1). The marked reduction of the descending phase by EGTA indicates that the late phase of BK-induced [Ca2+];increase is due to the influx of external Ca2+.These results demonstrated that BK-induced [Ca2+]i increases were produced not only by the internal Ca2+ mobilization but also by the influx of external Ca2+. Since the increase in the [Ca2+li level of the descending broad shoulder at 60 s after the stimulation was abolished by EGTA, it could be used as a parameter for Ca2+ influx (Fig. 3b). These suggestions appear to be supported by the data that

a. BK(200nM) g 600. E 400. 5 200. “m 2, t.

‘0

b. EGTA(O.SmM) +BK(200nM)

12

3 INCUBATION

4 TIME

5

6

7

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Fig. 1. Typical profiles of BK-induced calcium response. Normal human keratinocytes were triggered (a) by 200 nM BK, or (b) by BK in the presence of EGTA (0.5 mM).

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114

0

c. PMA+HJ+BK

2b). The peak [Ca*+]i at 20 s was slightly reduced to 294 * 149nM (n = 13cases) which was 83% of the control. However, there was no significant difference in the initial peak of [Ca*+]i at 20 s with or without the PMA pretreatment. On the other hand, the descendingphaseat 60 s was remarkably decreasedto 124 f 47 nM, 47% of the control (P < 0.01) (Figs. 2b and 3). In order to assesswhether PKC affects BKinduced Ca*+ responses, two inhibitors for the enzyme, H-7 and staurosporine, were used. As shown in Fig. 2, the suppressiveeffects of PMA on Ca*+ responseswere significantly alleviated by the

a) 0

Peak

[Ca*‘]i

‘zooH d. PMA+ST+BK

1000 t

.

.

INCUBATION TIME (mid

Fig. 2. Representative profiles of BK-induced calcium response. (a) The cells were stimulated by BK (200 nM) pretreated with (a) vehicle, (b) PMA (350 nM) for 20 min, (c) PMA (350 nM) + H-7 (50 PM) for 20 min, or (d) PMA (350 nM) + staurosporine (100 nM) for 20 min.

01

PMA

EGTA

PMA+H-7

PYA+ST

b) 60sec [Ca”]i 800 z 600E

the peak of Ins(1,4,5)P3 production induced by BK was seenat 20 s after the stimulation (Fig. 4). It is well known that phorbol esters directly activate PKC, and cause the translocation of PKC from cytosol to membrane in various types of cells including murine keratinocytes [ 10,111.In order to investigate the role of PKC in BK-induced [Ca*+]i responses, human keratinocytes were pretreated with PMA. When human keratinocytes were pretreated with PMA (350 nM) for 20 min, which is known to induce PKC translocation from cytosol to membrane, the shoulder of [Ca*+]i increase at 60 s was abolished and the descending phase of [Ca*+]i was significantly reduced (Fig.

control

0’

NS

, ,

***

control

* (

b

EGTA

PYA

**

, * ’

NS

PMA+H-7

1

PMAtST

Fig. 3. Characteristics of the BK-induced [Ca2+li responsesin normal human keratinocytes. The fura-24oaded cells were treated with EGTA (0.5 mM) for 5 min. PMA (350 nM), PMA (350 nM) + H-7 (50 PM) or PMA (350 nM) + staurosporine (100 nM) for 20 min, then stimulated by BK. Control was treated with BK alone. The first peak [Ca’+]i at 20 s after BKstimulation (a), and the second peak [Ca2+li at 60 s after BKstimulation(b) were determined. Each dot indicates the average of individual responded cells. Statistical analysis of the data was carried out by Student’s two-tailed unpaired I-test, *P < 0.01; **p< 0.005; ***p c 0.001.

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0 0

20

40

60

120

boo

INCUBATION TIME (SEC)

0

1

2

3

4

5

TIME (mid

Fig. 4. Time course of BK-induced Ins(l,4,5)Ps formation in keratinocytes. The keratinocytes were preincubated with 350 nM of PMA (0), 350nM of PMA + 50 PM of H-7 (o), or vehicle ( 0) for 20 min then stimulated with BK, or without stimulation (m) Each point represents the average of duplicate determinations.

Fig. 5. Time course of BK-induced [45Ca] uptake from external medium. The keratinocytes were preincubated with 350 nM of PMA (A) for 20 min, with 0.5 mM EGTA for 5 min (0) or with vehicle (O), and then stimulated with BK. Each point representsthe average of duplicate determination.

pretreatment with these two PKC inhibitors (Fig. 2). After pretreatment with H-7, the first peak of [Ca*+]i at 20 s was 356 * 189 nM (n = 14), and the shoulder at 60 s was 287 f 150 nM. In addition, the first peak [Ca*+]i at 20 s after pretreatment with staurosporine was 504 f 198 nM (n = 6), and the shoulder at 60 s was 389 f 124 nM. The descendingphasesof [Ca*+]i at 60 s after BK stimulation were enhanced by the pretreatment with PMA plus these PKC inhibitors compared to pretreatment with PMA alone (Fig. 3). There were no significant differences in [Ca*+]i increasebetween BK alone and BK with pretreatment of PKC inhibitors + PMA. These results strongly suggest that the suppressive effects of PMA on descending shoulder of BK-induced Ca*+ responsesare exerted through the action of PKC. BK caused Ins(1,4,5)Ps production peaking at 20 s (P < 0.001) compared with control, returning to the basal level at 60 s (Fig. 4). In addition, there were no statistically significant differences in Ins(1,4,5)PJ levels at 20 and 60 s among the cells treated with BK alone, BK + PMA and BK + PMA + H-7. From these findings, it was considered that the initial peak of [Ca*+]i rise was induced by Ins( 1,4,5)P3generation. The effect of

PKC on BK-induced [Ca*+]i rise might be limited to the descendingphase (shoulder) which was due to the external Ca*+ influx. In addition, BKinduced [4sCa] uptake was inhibited by preincubation with 0.5 mM EGTA for 5 min or 350 nM PMA for 20 min (Fig. 5). Talwar et al. [4] have shown that inositol trisphosphate was maximally increasedat 30 s after stimulation by 1 PM BK and remained elevated for at least 10 min, while Ins(1,4,5)PJ showed a transient increase in our study. This discrepancy may be due to differences in the methods used; we assayed Ins(1,4,5)Ps using a specific Ins( 1,4,5)Prbinding protein. The present investigation demonstrated that PMA, a well-known PKC activator, suppressed the descendingphase (shoulder) of BK stimulated Ca*+ responsesin human keratinocytes, and that the PKC inhibitors reversed the effects of PMA. These results suggest that PKC exerts a negative modulation for BK-induced Ca*+ increase. EGTA reduced markedly the descendingphase(shoulder) of the Ca*+ response, indicating that Ca*+ influx played a principal role in this phase. The Ca*+ profile in the presenceof PMA was similar to that after chelation of extracellular Ca*+ with EGTA. Thus, PKC was considered to affect at least in part Ca*+ influx from the extracellular medium. This

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notion was supported by the data that PKCinhibitors reversed PMA-induced suppression of Ca2+ influx. In PKC-down-regulated tibroblasts, enhanced and prolonged Ca2+ increases were observedupon agonist-stimulation by Corps et al. [ 121. They suggested that the prolonged Ca2+ responsescould be explained by decreasedCa2+ etlhtx due to PKC-down regulation. Furthermore, in NCB-20 neuroblastoma x Chinese hamster brain cell hybrid cells, it was reported that downregulation of PKC enhanced BK-induced phosphoinositide metabolism by relieving from the negative-feedback control operating between the receptor and PLC, and that the Ca2+ responses increased by accumulated Ins( 1,4,5)P, [ 131. To elucidate the mechanismsunderlying the negative modulation of PKC on Ca2+ influx, further studies will be required. References 111lnnis RB, Manning DC, Stewart JM, Snyder SH: [‘H]Bradykinin receptor binding in mammalian tissue membranes. Proc Nat1 Acad Sci USA 78: 2630-2634, 1981. PI Rosenbach T, Greenlee WF: Inositol phosphate formation in the human squamouscell carcinoma line SCC-12 F: studies with bradykinin, the calcium ionophore A23187, and sodium fluoride. J Invest Dermatol 96: 116-122, 1991. I31 RosenbachT, LiesegangC, Binting S, Czametzki M: Inositol phosphate formation and release of intracellular free calcium by bradykinin in HaCaT keratinocytes. Arch Dermatol Res 283: 393-396, 1993. [41 Talwar HS, Fisher GJ, Voorhees JJ: Bradykinin induces

phosphoinositide turnover, 1,2-diglyceride formation, and growth in cultured adult human keratinocytes. J Invest Dermatol 95: 705-710, 1990. 151Lee E, Yuspa SH: Changes in inositol phosphate metabolism are associated with terminal differentiation and neoplasia in mouse keratinocytes. Carcinogenesis 12: 1651-1658, 1991. 161Inohara S: Studiesand perspective of signal transduction in the skin. Exp Dermatol 1: 207-220, 1992. 171 Yuspa SH, Kilkenny AE, Steinert PM, Roop DR: Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentration in vitro. J Cell Biol 109: 1207-1217, 1989. 181Okano Y, Fu T, Nozawa Y: Calcium oscillation induced by bradykinin in polyoma middle T antigen-transformed NIH3T3 fibroblasts: evidence for dependenceon protein kinase C. Biochem Biophys Res Commun 176:8 13-819, 1991. [91 Seishima M, Takagi H, Okano Y, Mori S, Nozawa Y: Ganglioside induced terminal differentiation of human keratinccytes: early biochemical events in signal transduction. Arch Dermatol Res 285: 397-401, 1993. 1101Fu T, Okano Y, Hagiwara M, Hidaka H, Nozawa Y: Bradykinin induced translocation of protein kinase C in neuroblastoma NCB-20 cellDependence on 1,2diacylglycerol content and free calcium. Biochem Biophys Res Commun 162: 1279-1286, 1989. 1111Chida K, Kato N, Kuroki T: Down regulation of phorbol diester receptors by proteolytic degradation of protein kinase C in a cultured cell line of fetal rat skin keratinocytes. J Biol Chem 261: 13013-13018, 1986. WI Corps AN, Cheek TR, Moreton RB, Berridge MJ, Brown KD: Single-cell analysis of the mitogen-induced calcium responses of normal and protein kinase Cdepleted Swiss 3T3 cells. Cell Regul 1: 75-86, 1989. iI31 Okano Y, Higashida H, Fu T, Sakai T, Nozawa Y: Bradykinin induced phosphoinositide dependent responsesin protein kinase C down-regulated NCB 20 cells. Neurochem Int 18: 419-424, 1991.