Vol. 143, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS March 13, 1987 Pages 539-544

POSSIBLE INVOLVEMENT OF PROTEIN’KINASE C IN PARATHYROID HORMONE DEGRADATION BY OSTEOBLAST-LIKE t?AT OSTEOSARCOMA CELL LLNE UMRIOG

‘Toru Yamaquc:h i * ,Hisamitsu Raba,Masaaki Fukase. Yoshikazu liinoshita,Tadao Fujimi,and Takuo Fujita

Third Division, Dt:parl.ment of Medicine, Kobe University School of Medi tine.

7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650, Japan

Received January 27, 1987

Summary : The effects of I;!-0-t.etraade~anoyl ~ho1~l~ol-13-ace- tate (TPA).I-oleoyl-2-acet,yl-cjlycerol(OAG).arld I-(5-isoquinoline- sulfonyl )-2-methylpiperazine (H-7) on the parathyroid hormone (PTH) deqradinq activity in a PTH-responsive osteoblast-like rat

line LIMRIOG were invest.iqated to ~~~~os~~co~~2+C~~~:ivatrd assess the _ . phosphol ipid dependent protein kinase

(protein kinase C) on the deqradat ion of hormones. TPA and 0AG , act. i va tors of protein kinase C, enhanced the PTH desrad i ny activity dose-dependently. whereas H-7. an inhibitor of protein kinase C. exhibited a dose-dependent inhibition on this activity. These data suggest that protein kinase C activation may enhance PTH deqradinq activity by UMRlO6 cells as a possible reyulator 01 PTH deqradat ion. 0 1987 Academic Press, Inc.

caz+ -activated. phosphotipid dependent protein kinase (pro-

tein kinase Cl. an enzyme which is ubiquitous in tissue and

organs (1) and activated by the receptor-mediated hydrolysis of

inositol phospholipids (2). plays a crucial role in transduci nq

various extracellular siqnals into the cell (3). Protein kinase C

miqht also be involved in the release of hormones. such as insu-

lin (4). aldosterone 15). calcitonin 16). somatostatin (7). and

parathyroid hormone (PTH) (8). but it still remains unclear

whether or not this enzyme is concerned in the deqradat ion of

hormones.

PTH is known to be degraded in the parathyroid gland (9).

liver (10) and kidney (11.121. In previous work (131, we showed

To whom correspondence should be addressed.

0006-291X/87 $1.50

539 Copyright 0 I987 by Academic Press. Inc.

All rbhts of reproduction in any ,form reserved.

Vol. 143, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the existance of a limited PTH-hydrolyzing activity in a osteo-

blast-like rat osteosarcoma cell line UMR106 (141, and suggested

the participation of osteoblasts in the degradation of PTH.

In the present study, using this cell line. the ef fect,s of

protein kinase C activators. 12-0-tetraadecanoyl phorbol-13-

acetate (TPA) (15.1G) and I-oleoyl-2-acctyl-glycerol (OAG) 1171.

and a protein kinase C inhibitor. I-c5-i.soquinolinesulfonyl~-2-

methylpiperazine (H-7) (18-20). on the PTH degradi rig activity

were examined to clarify a role of prot,ein kinase C on this

mechanism.

Materials and Methods

Materials: Human I)TH- ( 1-84 1 was purchased from Pept. ide Ins t i tut e Inc. (Osaka.JAPANJ. TPA from Sigma Chemicals Co. (St.Louis.MOJ. OAG from Avanti Polar-Lipids.Inc. (Birmingham.AL). H-7 and N-(2- guanidinoethylJ-5-isoguinolinesulfonamide hydrochloride (HA10041 from Seikagaku Kogyo Co.Ltd. (Tokuo.JAPAN1.

Cell culture: UMRIOG cells were kindly donated from Dr.T.J.Martin (University of Melbourne. Australia). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEMI containing 10 % fetal calf serum in a humidified 95 % air/5 % CO2 at 37 C.

Measurement of PTH fragments degraded from hPTH-(l-84) by UMR106 cells: After reaching6confIuence. the mgnolayers of UMR106 cells (approximately 4.0X10 cells per 9.6 cm” dish) were preincubated in the 1.5 ml of experimental medium (serum free DMEM) containing test agents or vehicle alone at :37 C for 60 min. Then, 75 r1g

hPTH-(l-84) in 25 pl of 0.01 N HCI was added into the medium and the cells were further incubated for 60 min. For the control study. after 60 min preincubation of the cells with 1.5 ml of med i urn containing 50 rig/ml TPA. 1 ml aliquot of medium was re- moved and the medium was incubated in the presence of 50 ng hPTH- (l-84) for 60 min. As previously described (13). PTH fragments in the medium. degraded from hPTH-(l-84) by UMR106 cells during 60 min. were separated from intact PTH by adding trichroloacetic acid and bovine serum albumin to yield final concentrat.ions of 5 % and 0.25 %. respectively. They were measured by radioimmuno- assay using a Yamasa PTH-RIA kit (Yamasa Shoyu Co.Ltd. Choshi.JA- PAN).

Statistical analysis: The results were expressed as the mean +SEM. Statistical significance was assessed by Student’s t-test.

Results

Dose-dependency of TPA on the PTH degrading activity in the

ccl 1s during 60 mi II incubation was shown in I:ig 1. TPA at the

540

Vol. 143, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0’ I ’ ’ 0 1 0 10 TPA 20 (ns/mQ) 50 0 2 0 10 0% bdmQ) 50 100

Figure 1. Dose dependent effect of TPA on the PTH degrading activity in UMR106 cells. The iPTH fragments’ production was significantly increased by TPA. Vertical lines represent the mean +SEM of 3 different cultures. * p<o.o5: significantly different from the value obtained in the absence of TPA.

Figure 2. Dose dependent effect of OAG on the PTH degrading activity in UMRlO6 cells. The iPTH fragments’ production was significantly increased by OAG. Vertical 1 ines represent the mean+SEM of 5 different cultures. ** P<0.001: significantly dif- ferent from the value obtained in the absence of OAG.

concentration of 20 rig/ml stimulated the product. i on of 1 inmuno-

reactive PTH (iPTH) fragments significantly. and raised it to 254

% over t,he value obtained in the absence of TPA. al 50 VJ/l?ll

1P<O.O5). On the contrary. no significant PTH degrading activity

was found in the cont,rol st,udy (data not shown).

Synthetic diacylglycerol, OAG, also stimulated the PTH deg-

radat ion dose-dependently as shown in Fig 2. In the presence of

OAG at the concentrations of 50 pg/ml and 100 pg/ml. the produc-

tion of iPTH fragments increased significantly up to 125 % and

129 % of the value obtained in the absence of OAG. respectively

(P<O.OOl 1.

FicJ :< showed the individual inhibitory effect of H-7. a

potent inhibitor of Protein kinase C, and that of HA1004, a weak

inhibitor of this enzyme (20.211 on the PTH degrading activity in

the cells. H-7 at the concentrat.ion of 20 pM inhibited t,he iPTH

fragmentation s i gn i f i cant 1 y (29 % inhibition of the value ob-

tained in the absence of H-7. P<O.O5)and dose-dependent.ly inhi-

bited it by 75 % at 200 pM (P<O.OOl). On the other hand. no

541

Vol. 143, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Ii7 or HA1004(/1M)

Figure 3. Dose dependent effects of H-7 and HA1004 on the PTH degrading activity in UMRlO6 cells. H-7 inhibited the i PTH fragments’ production strongly, whereas HA1004 inhibited it only slightly. Vertical lines represent the mean +SEM of 3 different cultures. * p<o.o5: ** p<o.o01: significantly different from the value obtained in the absence of H-7 or HA1004. C-1 :H-7, to---o):HA1004.

inhibitory effect of PTH fragmentation was observed by HA1004 at

20 PM and 50 pM and was only 20 % even at the concentration of

200 pM.

Discussion

The possible involvement of protein kinase C in the

degradation of hormones was first demonstrated using a

osteoblast-like rat osteosarcoma cell line UMRlO6 which has a

, limited PTH-hydrolyzing activity (13).

This ccl 1 1 ine is known to respond to PTH and produce adeno-

s i ne-cycl i c 3 ’ ,5 ’ -monophosphat.e (CAMP) as a second messenger

(14). and cyclic AMP subsequently activates CAMP dependent pro-

tein kinase (protein kinase A). In addition, Lerner U.H. et al.

recently reported t.hat PTH provoked the hydrolysis of inosi to1

phosphol ipids in UMHlO6 cells (22). One of their products. di-

acylglycerol. activates protein kinase C (2.31, and the other,

inositol-1.4.5-triphosphate (I-1.4,5-P31. serves as a medi at~or of

Ca2+ mobilization from an internal store (231. These findings

542

Vol. 143, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

indicate that in LlMRlO6 cells, signals from PTH are transduced

into the ccl 1 via at least t,hree pathways; those mediated by

protein kinase A and protein kinase C and the Ca2+ signaling

pathway.

Tumor-promoting phorbol esters, such as TPA. have a

structure very similiar to diacylglycerol and activate protein

kinase C directly as its substitute (15,161. Several 1 i nes 0 f

evidence support the notion that protein kinase C itself is the

receptor of phorbol esters (24 1. Some synthetic diacylglycerols,

such as OAG, are readily intercalated into intact cell membranes

and also activate protein kinase C directly (17). We have demon-

strated that these Protein kinase C activators, TPA and OAG,

enhanced the PTH degrading actrvity in lJMR106 cells dose-depen-

dently. Additionally. we have examined the ef fect,s of H-7 and

HA1004. newly synthetized compounds related to isoquinoline-

sulfonamides 118-211, on this degrading activity. H-7 is known to

be a potent inhibitor of protein kinase C (Ki value is 6.0 PM).

whereas HA1 004 is a weak inhibitor of this enzyme (Ki value is

40.0 pM) (20). In our study. the PTH degrading activity in UMRlO6

cells was strongly suppressed by H-7 but. only weakly inhibited by

a large amotlnt of HA1004 (200 PM). All these findings strongly

suggest that protein kinase C activation is involved in the

enhancement of PTH degrading activity in UMRlU6 cells. tlowever .

HA1004. which is a potent inhibitor of protein kinase A (Ki value

is 2.3 PM) (20) gave no influence on the PTH degrading activity

at the concentrations of 20 pM and 50 PM. suggesting that protein

kinase A might not play a crucial role in the PTH degrading

mechanism in this cell. We are now further investigating whether

2+ or not protein kinase A and the Ca signaling pathway have any

involvement in the PTH degrading activity in this PTH-respons i ve

cell line.

543

Vol. 143, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

J n summary. the present data suggest that protein kinase C

might be involved in the PTH degrading activity in UMR106 cells

and could become a new candidate for a regulator of the

degradation of hormones.

Acknow 1 edsmen t

We are thankful to Yamasa Shoyu Co.Ltd. for supplyins us with PTH-RJA kits.

1 ,

2. 3. 4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21. 22.

23. 24.

References

Kuo,J.F..Anderson,R.G.G..Wise,B.C..Macke~~lova,L..Salomonsson, I..Brackett.N.L..Katoh,N.,Shoji.M., and Wrenn,R.W. (1980) Proc.Natl.Acad.Sci.USA 77.7036-7043. Nishizi1ka.Y. (1984) Nature 308.693-698. Nishi2uka.Y. (1986) Science 233.305-312. Znwa1ich.W. .Brown.C.. and Rasmussen,H. (1983) Biorhem.Biophys. Res.Commun. 117,448-455. Ko.jima, I . ,Lippes.H. .Ko.jima.K., and Rasmussen,H. (19831 Biochem.Biophys.Res.Commun. 116.555-562. Hishikawa,R.,Fukase,M..Yamatani,T..Kadowaki,S,, and Fujita.T. (19851 Biochem.Biophys.Res.Commun. 132.424-429. Yamatani,T.,Kadowaki,S..Chiha,T..Chihara,K..Fukase.M., and Fu,iita.T. (1985) Life Sci. 37,2415-2419. Brown.E.M.,Redsrave,J., and Thatcher,J. (1984) FEBS lett. 175,72-75. Hamilton,J.W.,Jilka.R.L.. and MacGre9or.R.R. (1983) Endocrinology 113.285-292. Segre,G.V., Perkins,A.S..Witters.L.A., and P0tts.J.T. 11981) J.Clin.Invest. 67,449-457. Fujita.T.,Ohata,M.. and Yo5hikawa.M. (19691 Endocrinol.Japon. 16,383-389. Botti,R.E.,Heath.E.,Frelinser.A.L.,Chuang.J..Roos.B.A., and Zul1,J.E. (1981 ) J.Biol.Chem. 256.11483-l 1488. Yamaguchi.T., Baba,H..Fukase.M.,Kinoshita,Y.,Fujimi,T.. and Fu.jita,T. (1986) Biochem.Biophys.Hes.Commun. 141.762-768. Martin.T. J., Insleton,P.M.,Underwood.J.C.E.,Michellangelli. V.P. 1 and Hunt.N.H. (1976) Nature 260,436-438. Castasna.M.,Takai,Y..Kaibuchi,K.,Sano,K.,Kikkawa,U., and Nishizuka.Y. (1982) J.Biol.Chem. 257.7847-7851. Yamanishi,J.,Takai,Y..Kaibuchi,K.,Sano.K..Castagna,M,, and Nishizuka,Y. (1983) Biochem.Biophys.Res.Commun. 112.778-786. Kaibuchi,K.,Takai.Y.,Sawamura,M.,Hoshijima,M.,Fujikura,T., and Ni5hizuka.Y. (1983) J.Biol.Chem. 258.6701-6704. Inasaki,M.,Kawamoto,S., and Hidaka,H. (1984) J.Biol.Chem. 259.14321-14323. Kawamoto,S.. and Hidaka,H. (1984) Biochem.Biophys.Res.Commun. 125,258-264. Hidaka.H.,Inasaki,M.,Kawamoto.S.. and Sasaki,Y. (1984) Biochemistry 23.5036-5041. Asano,T., and Hidaka,H. (1984) J.Pharmacol.Exp.Ther. 231,141-145 Lerner.U.H..Sahlberg,K.. and Fredholm,B.B. (1986) IXth International Conference on Calcium Regulating Hormones and Bone Metabolism,Nice,France. Abstract No.28. Berridse.M.J., and Irvine,R.F. (1984) Nature 312.315-321. Niedel.J.M.,Kuhn,L.J., and Vanderbark,G.R. (1983) Proc.Natl.Acad.Sci.USA 80.36-40.

544