Biochimica et Biophysica Acta. 1074(1991) 182-188 1991 ElsevierSciencePublishersB.V.0304-4165/91/$03.50 ADONIS 0304416591001746


BBAGEN 23515

The effect of PGF2~ on parathyroid hormone-stimulated cyclic A M P production in mouse osteoblastic cell, MC3T3E 1 Seiki W a d a , Y o s h i r o u Y a s u t o m o , H i r o s h i K o s a n o , N o b u o K u g a i a n d N a o k a z u N a g a t a The Third Department of Internal Medicine. National Defense Medical College. Tokorozawa. Saitama (Japan)

(Received27 December1990) Key words: Prostaglandinlea; Parathyroidhormone;ProteinkinaseC; cyclicAMP; Osteoblasticcell. MC3T3EI; (Mouse) The effect of prostaglandin Fza (PGF2a) was investigated in MC3T3Et cells on the succeeding cAMP response to parathyroid hormone (PTH). PGF2~ increased the membrane.associated protein kinase C (PKC) activity, indicating the activation of this enzyme. The effect of PTH to increase cAMP production was enhanced by pretreatment with PGF2a. Phorbol 12-myristate 13-acetate also enhanced cAMP production stimulated by PTH, and PKC inhibitor H7 attenuated the enhancement of PGF2a. A23187 did no~. reproduce the PGF2,, effect, and this effect was not antagonized by the calmodulin antagonist W7. PGFza did not change the EDso nor the maximally responsive dose of PTH in stimulating cAMP production. The effect of PGF2a was not affected by pertussis toxin, and PGFza also enhanced cholera toxin- or forskolin-stimulated cAMP production. In accordance with the response of cAMP to PTH, the resorption of mouse limb bones stimulated submaximaily by PTH was enhanced by the ccacomitant presence of PGFz,. These results indicate that PGF2~ modulates cAMP response through the activation of PKC, the target of which might be the catalytic unit of adenylate cyclase. Such interaction between signal transduction systems may have significance in modulating the effect of PTH on bone, i.e., bone resorption. Introduction Activation of adenylate cyelase and phosphatidyl inositol (PI) turnover along with a change of intracellular calcium concentration has been accepted as a major ubiquitous signal transduction mechanism. In the clonal mouse osteoblastic cell MC3T3Et, PTH, a most important hormonal regulator of bone metabolism, is known to stimulate cyclic AMP (cAMP) production and activate cAMP-dependent protein kinase, with a minor effect on PI turnover [1]. On the other hand, prostaglandin F2~ (PGF2~), which is a product of bone cell [2] and apparently plays a role as a local regulator of bone remodeling, stimulates PI turnover and increases intracellular calcium concentration with little effect on cAMP production [3-5]. Abbreviations: H7, l-(5-isoquinolinesulfonyl)-2-methylpiperazinedihydrochloride; HA1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrocbloride; W7, N-(6-aminohexyl)-5-cbloro-l-naphthalenesulf0namide hydrochloride; PI, phosphatidylinositol; PTH, parathyroid hormone;PGF2,, prostaglandinF2a; PKC, protein kinase C. Correspondence:S. Wada, The Third D,~par|,nen!of Inlernal Medicine, National Defense MedicalCollege,Tokorozawa, Saitama 359, Japan.

Recently. the interaction between these second messengers has been examined in several systems, and either attenuation or potentiation of adenylate cyclase response is observed by the activation of PI turnover or an increase of intracellular calcium concentration [6-14]. The presence of such cross-talk among these messengers may have general significance in modulating a signal transductlon mechanism. In the present study we used MC3T3E, cells in culture to examine the effect of PGF2~ on the succeeding response of cAMP to PTH. and also to study the effect of PGF2~ on resorption of mouse limb bone stimulated by PTH. Although PGF2, has been shown to stimulate PI turnover [4,5], direct evidence that PKC is actually activated is lacking. Therefore we first examined the effect of PGF,,, on the distribution of PKC between the cytosol and membrane fractions. The translocation of the enzyme activity to membrane fraction is known to indicate the activation of the enzyme [15-20]. Materials and Methods Materials

PGF2~, phorbol 12-myristate 13-acetate (PMA), phorbol 12-monoacetate, phorbol 12,13-didecanoate, 4a-phorbol 12,13-didecanoate, A23187, bovine serum

183 albumin (BSA). phenylsulfonylfluoride. Nonidet P-40. actinomycin D, cholera toxin, and pertussis toxin were obtained from Sigma Chemicals (St. Louis, MO), Iorsko!in was from Calbiochem (La Jolla, CA); a-modified minimum essential medium (a-MEM) and fetal bovine serum (FBS) were from M.A. Bioproducts (Walkersville, MD): Eagle's minimum essential medium (MEM) was from Nissui Pharmaccutical~ (Tol,_yu. Japan): H7. HAl004, and W7 were purchased from Seikagaku Kc,gyo (Tokyo, Japan). 3-1sobutyl-l-methyl-xanthine (IBMX) and ~:enzamidine hydrochloride hydrate were from Aldrich Chemicals (Milwaukee, WI); plastic tissue culture dishes were from Costar (Cambridge, MA) and Falcon Plastics (Los Angeles, CA); and synthetic human (h) PTH(1-34) was donated by Toyo Jyozo (Tokyo. Japan). The mouse osteoblastic cell (MC3T3EI) was generously presented by Professor H. Kodama (Ohu Dental University). The cAMP assay kit and protein kinase-C (PKC) enzyme assay kit were purchased from Yamasa Shoyu (Chiba, Japan) and Amersham International (U.K.), respectively.

Cell culture The cells were maintained in a - M E M containing 10% FBS in 95% air, 5% CO, incubator at 37 ° C. and were subcultured every 3 days. Determination of cA MP response The cells (2.104) were cultured in 10 m m multiplate culture dishes in 0.5 ml a - M E M containing 10% FBS for 4 days, and variously pretreated as indicated in each experiment. To examine the response to PTH, cholera toxin, isoproterenol, or forskolin, the medium was replaced with fresh FBS-free medium containing 0.1% BSA and 2 m M IBMX. After 10 min treatment, cellular cAMP was extracted by sonicating for 10 s in 0.1 M HCI. cAMP was measured with a Yamasa RIA kit. PKC activity determination The cells were cultured for 4 days in 90 m m dishes in a - M E M containing 10~ FBS. After the medium was replaced with FBS-free a - M E M containing 0.1% BSA, either PIJF2,,, PMA, or PFH was added and the cells were incubated further for the time indicated. After the medium was discarded, the cells were washed twice with 10 ml ice-cold PBS. The cells were scraped using a rubber policeman with 1 ml of buffer A (20 m M TrisHCI (pH 7.5), containing 0.25 M sucrose, 5 m M EDTA, 10 m M EGTA, 50 / t g / m l phenylmethylsulfonyl fluoride, 10 m M benzamidine, 0.3% v / v B-mercaptoethanol), sonicated for 30 s, and then centrifuged at 1 0 0 0 0 0 × g for 60 rain at 40C. The pellet was resonicated ia buffer A containing 1~ Nonidet P-40 for 20 s, and left for 60 n'fin at 4 " C . Separate aliquots from the supernatant (cytosolic fraction) and the resonicated pellet (membrane fraction) were assayed for PKC activ-

ity by Amersham's PKC enzyme assay kit [21]. Data are expressed as PKC specific activity (pmol transferred phosphate per rain per mg total protein). The concentration of total protein in the dishes was determined by the method of Lowry et al. [22], with bovine serum albumin as standard.

Bone resorption was measured according to the method described by Sato et al. [23]. Fetal mouse limb bones were labeled with ":~Ca by mjecting the mother with 2MBq ~SCaCI., (Amersham) on the 15th day of gestation. Labeled bones were precuhured for 1 day to remove freely exchangeable 45,Ca and transferred to fresh MEM with agonists and cultured for an additional 72 h. Resorption was assessed as the percentage of the total '~SCa that was released into the medium during the 72 h treatment period. Total bone radioactivity was calculated as the sum of radioactivity released into the medium plus radioactivity in the 10% (v/v) trichloroacetic acid soluble mineral fraction of the bone after culture.

Statistical attalysis The Student t-test was used to evaluate the data obtained in these experiments. A

:tyR.NE TIME (rain) ~1°°1 B


TIME (rain) Fig. l. Time-courseof PGF2aeffect on protein kinase C activity in MC3T3EI cells.Cells (5-106) were incubatedwithout (control: - o -) or with PGF~, (10-v M; -e-), and membrane (A) and cytosol (B) fractions were assayed for protein kinase C. Each point shows the mean± S.D. for three dishes. * p < 0.O5: * * P

The effect of PGF2 alpha on parathyroid hormone-stimulated cyclic AMP production in mouse osteoblastic cell, MC3T3E1.

The effect of prostaglandin F2 alpha (PGF2 alpha) was investigated in MC3T3E1 cells on the succeeding cAMP response to parathyroid hormone (PTH). PGF2...
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