Inyolvement of Protein Kinase C in the Regulation of Cortisol Production by Guinea Pig Adrenocortical Cells

Summary Cytosol of the guinea pig adrenals was found to contain a protein kinase which was dependent on the presence of both calcium and phospholipids (phosphatidylserine and diolein), i. e., calcium/phospholipid-dependent protein kinase (protein kinase C). The peak of protein kinase C was separated from type II cAMP-dependent protein kinase by DE-52 chromatography. 12-0-Tetradecanoylphorbol-13-acetate (TPA) caused dose-dependent increments of Cortisol formation without affecting cAMP formation by guinea pig adrenocortical cells as well as angiotensin II did. TPA-activated Cortisol production was blocked by the addition of aminoglutethimide and cycloheximide, suggesting that the site of action of TPA might be located at a point before the production of pregnenolone in the mitochondria. Since TPA showed an increase in the Cortisol production, protein kinase C may be involved in modulating steroidogenesis in the guinea pig adrenals in addition to the classical cAMP-dependent protein kinase pathway.

Ca + 2 /phospholipid-dependent protein kinase (protein kinase C) is reported to be activated by calcium and to be present in various tissues, suggesting that protein kinase C may play a crucial role on hormone action as an intracellular signal (Minakuchi, Takai, Yu and Nishizuka 1981). Phorbol esters which can activate protein kinase C have been shown to modulate the Cortisol production by bovine adrenocortical cells (Vilgrain, Cochet and Chambaz 1984; Finn, Stehle, Ricci and Hofmann 1988). However, there is no information about protein kinase C and its relationship to steroidogenesis in guinea pig adrenals which produce Cortisol, as do the adrenals of human beings. Thus we tried to compare the effect of angiotensin II which was reported to activate phosphoinositide turnover in bovine adrenocortical fasciculata cells (Hadjian, Guidicelli and Chambaz 1982; Hadjian, Culty and Chambaz 1984a; Hadjian, Culty and Chambaz 1984b) on cAMP formation and Cortisol production by cells isolated from guinea pig adrenals with that of TPA in order to clarify precisely the action of TPA on the regulation of steroidogenesis. These experiments were therefore performed in order to investigate the role of protein kinase C on the regulation of Cortisol biosynthesis in guinea pig adrenals.

Key words TPA - Protein Kinase C - Adrenocortical Cells - Steroidogenesis - Guinea Pig

Introduction It has become increasingly apparent that both cAMP and calcium play crucial roles as intracellular second messengers in mediating the action of hormones. In adrenocortical cells Cortisol production is mainly regulated by cAMP and cAMP-dependent protein kinase (Halkerston 1975). However, it was reported that angiotensin II stimulates Cortisol production in bovine fasciculata cells by a cAMP-independent mechanism (Vallotton, Capponi, Grillet, Knupfer, Hepp, Khosla and Bumpus 1981), and calcium plays an important role in the regulation of corticosterone formation as well as the cAMP-dependent process does (Podesta, Milani, Steffan and Neher 1980).

Horm. metabol. Res. 22 (1990) 29-32 © GeorgThiemeVerlagStuttgart • New York

Materials and Methods Collagenase, deoxyribonuclease I (DNase, bovine pancreas), cAMP, BSA (fraction V), and histone HA were purchased from Sigma Chemical Co. (St Louis, MO). Histone HI and 12-0-tetradecanoylphorbol-13-acetate (TPA) were obtained from Funakoshi Pharmacological Co. (Tokyo, Japan). [y-32P]-ATP was purchased from New England Nuclear, Boston, MA. Organic solvents and other chemicals were used as obtained from suppliers. Male guinea pigs (Hartley strain), weighing approximately 800 g, were obtained from Takasugi Animal Co. (Saitama, Japan), and killed without stress by decapitation at between 9:30 and 10 AM. Five or six animals were generally used for each experiment. The adrenals were quickly removed and placed in MEM at room temperature, and zona fasciculata which can mainly produce Cortisol was obtained as previously reported (Nishikawa and Strott 1984). The portion of zona fasciculata was minced with scissors and placed in MEM containing 5 mg/ml collagenase, 0.1 mg/ml DNase, and 0.2% BSA. The isolated cells were prepared as previously reported (Nishikawa and Strott 1984). The cells were suspended in MEM-0.2% BSA to attain a final concentration of 1 x 105 cells/ml. Cell viability as determined by trypan blue exclusion was 92.6 ± 7.6 %. Cell suspensions

Received: 6 Dec. 1988

Accepted: 5 Apr. 1989

Downloaded by: NYU. Copyrighted material.

T. Nishikawa, Akiko Yoshida, Y. Tamura and S. Yoshida Department of Internal Medicine (II), School of Medicine, Chiba University, Chiba City, Chiba, Japan

Horm. metabol. Res. 22(1990)

T. Nishikawa, Akiko Yoshida, Y. Tamura andS. Yoshida Fig. 1 DE-52 chromatography. Theguinea pig adrenals were homogenized in 10 mM potassium phosphate buffer, pH 6.8,0.25 M sucrose, 2 mM EDTA, 0.5 mM MIX, and 6 mM 2-mercaptoethanol. The homogenate was centrifuged at 105,000 x g, for 60 min and the supernatant was then applied toa0.9 x 4 cm DE-52 column. The column was eluted with 0-0.3 M NaCI in 20 mM Tris-HCI, pH 7.4,0.25 M sucrose, 5 mM 2-mercaptoethanol, and 2 mM EDTA, and fractions of 1 ml were collected. An appropriate amount of each fraction was used for estimating protein kinase C activity and protein kinase A activity. Each kinase activity was determined as described in Materials and Methods.

Downloaded by: NYU. Copyrighted material.

30

Fig. 2 Effects of TPA and angiotensin II on Cortisol production. Cells isolated from the guinea pig adrenals were incubated as described in Materials and Methods. TPA and 4a-PDD was dissolved in acetone and added to the incubation medium in afinal concentration of 0.2% acetone. Control incubation tubes also had 0.2 % acetone which did not affect the cell viability.

were preincubated for 30 min at 37 °C in an atmosphere of 95 % O25 % CO2 in a humidified chamber. The cells were pelleted and resuspended in 2 ml of fresh MEM-0.2 % BS A with and without specific additives. Incubations were performed at 37 °C in 95 % 02-5 % CO2 for2 hrs and terminated by transferring the tubes to an ice bath. The cells were pelleted and the supernatant was removed and stored at -20 °C until analyzed. Cell viability was again checked by the trypan blue exclusion test at the end of the 2-hr incubation period and was found to be 91.4 + 4.6%. For the routine determination of Cortisol, 200-u.l aliquots of incubation medium were extracted with dichloromethane, and Cortisol was assayed by radioimmunoassay without further purification. Isolated cells were incubated in MEM containing 0.2% BSA and 0.2 mM l-methyl-3-isobutylxanthine (MIX) after preincubation which was performed as described above. Incubation was terminated by adding 200 ul of 30 % TCA followed by sonication. The samples were centrifuged and supernatant was treated four times with cold diethyl ether. cAMP was estimated by radioimmunoassay, according to the method of Honma, Satoh, Takezawaand W(1977).

Fig. 3 Effects of TPA and angiotensin II on cAMP formation. Upper panel shows the effects of 10~° M TPA and 10" 6 M angiotensin 11 on cAMP formation during 5,15, and 30-min incubations. Lower panel depicts the effect of various concentrations of TPA and angiotensin II on cAMPformation when incubated for 5 min. Results are the mean ± SE of three or four separate experiments, each performed in triplicate.

The activities of protein kinase C and protein kinase A were determined by the methods of Kikkawa, Minakuchi, Takaiand Nishizuka (1983), and Roskoski (1983), respectively. Protein was measured by the method of Lowry, Rosebrough, Fair and Randall (1951). All results represent at least three separate experiments, each performed in more than triplicate. The results were expressed as the mean ± SE. Statistical evaluation was done by analysis of variance, followed by Student's t test. Results were considered significant at p < 0.05.

Horm. metabol. Res. 22 (1990) 31

Table 1 The effect of aminoglutethimide and cycloheximide on TPA-activated Cortisol production Additives

Cortisol production (% control)

no addition TPA,10" 6 M aminoglutethimide, 10" 4 M cycloheximide, 10 u.g/ml TPA, 10~6 M + aminoglutethimide, 10" 4 M TPA, 10~6 M + cycloheximide, 10 ng/ml

100 ± 227 ± 88 ± 105 ± 79 ± 118 ±

9 15a 3 17 19 b 7°

Isolated adrenocortical cells (105 cells/ml) were incubated with or without TPA and various substances, and Cortisol levels were determined after 2 h of incubation as described in Materials and Methods. Results are the mean ± SE of three orfour separate experiments, each performed in triplicate. The basal production of Cortisol was 5.5 ± 0.5 ng/105 cells. a p < 0.001 compared with no addition b p < 0.001 compared with 10" 6 MTPA c p < 0.01 compared with 10" 6 MTPA

Results

cAMP-independent mechanism. It was also reported that angiotensin II increases Cortisol production by activating phosphoinositide turnover with possible enhancing protein kinase C in bovine adrenocortical cells of fasciculata origin (Hadjian, Guidicelli and Chambaz 1982; Hadjian, Culty and Chambaz 1984a; Hadjian, Culty and Chambaz 1984b). The present investigation clearly demonstrated that TPA stimulated Cortisol production in a dose-dependent manner in cells isolated from guinea pig adrenals as well as angiotensin II did. Since no alteration in the level of cAMP was observed with various concentrations of TPA and angiotensin II under the condition where phosphodiesterase was inhibited by MIX, it appears that TPA stimulates Cortisol production in guinea pig adrenals by a cAMP-independent mechanism as well as angiotensin II does. It was reported that steroidogenesis is stimulated by TPA without increasing the level of cAMP in bovine adrenocortical cells (Culty, Vilgrain and Chambaz 1984). Thus the present data concerning the effect of TPA on cAMP formation by cells isolated from guinea pig adrenals are consistent with the previous findings reported by Culty, Vilgrain and Chambaz (1984).

It has recently been reported that Cortisol formation from bovine adrenocortical fasciculata was regulated by protein kinase C system (Vilgrain, Cochet and Chambaz 1984; Kenyon, Anyaorah, Woodburn, Connell and Fraser 1988). These reports are consistent with the present experiments using guinea pig adrenals. Thus, the activity of an adrenal cell producing Cortisol is controlled by an intricate network of intracellular signals of which cAMP and calcium are key components. It is very important to investigate which As shown in Fig. 2, TPA activated Cortisol pro- step(s) of steroidogenesis are activated by protein kinase C. duction in a dose dependent manner with a maximally active Aminoglutethimide and cycloheximide have been reported to concentration of 10"6 M. 4a-phorbol 12,13-didecanoate (4a- interfere with the conversion of cholesterol to pregnenolone PDD) which is not active for protein kinase C did not affect the by inhibiting the cholesterol side-chain cleavage which is a Cortisol formation. Fig. 2 also shows that angiotensin II stimu- rate-limiting step of adrenal steroidogenesis (Uzquiris, lated Cortisol production as well as TPA did. The effects of Whipple and Salhanick 1977; Simpson 1979). These two inhibTPA and angiotensin II on cAMP formation are shown in Fig. itors clearly induced an inhibition in TPA-activated Cortisol 6 6 3. As shown in Fig. 3, neither 10~ M TPA nor 10~ M angi- production. These results indicate that activation of protein kiotensin II changed the cAMP formation when incubated for 5, nase C by TPA may stimulate the cholesterol side-chain cleav15, and 30 min. Various concentrations of TPA and angioten- age and/or steroidogenic steps before the cholesterol sidesin II did not affect the cAMP formation during 5-min incuba- chain cleavage. It was reported that P-450 in bovine tion. adrenocortical mitochondria was selectively phosphorylated by protein kinase C, although it is not known yet whether such The effect of aminoglutethimide, and cyclo- phosphorylation leads to a modulation of cholesterol sideheximide on TPA-activated Cortisol production is shown in chain cleavage enzymes (Vilgrain, Defaye and Chambaz Table 1. TPA-activated Cortisol production was inhibited by 1984). From the results of the effect of 25-hydroxycholesterol adding aminoglutethimide and cycloheximide. on luteal steroidogenesis, Brunswig, Mukhopadhyay, Budnik, Bohnet and Leidenberger (1986) postulated the possibilities of TPA-increased availability of cholesterol to the mitochondrial Discussion side-chain cleavage enzymes and/or TPA-activated affinity of The present experiments demonstrated that the enzymes for the substrate. Moreover it was reported that the guinea pig adrenocortical tissue possesses the Ca/phos- the Gi inhibitory components of the adrenocortical cell adenypholipid-dependent protein kinase originally found in brain late cyclase system may be inactivated by TPA, leading to intissue by Nishizuka and coworkers (Takai, Kishimoto, Inoue creased response to ACTH (Brami, Vilgrain and Chambaz and Nishizuka 1977). Protein kinase C is reported to be present 1987). Thus it was suggested that protein kinase C-dependent in various tissues in mammals (Minakuchi, Takai, Yu and process may be involved in the regulation of cell membrane Nishizuka 1981), including steroid-secreting tissues (Kojima, functions including the adenylate cyclase system. It is specuKojima, Kreutter and Rasmussen 1984; Vilgrain, Cochet and lated from the present data and the previous findings that actiChambaz 1984). Castagna, Takai, Kaibuchi, Sano, Kikkawa vation of protein kinase C may act on early step(s) of guinea and Nishizuka (1982) postulated that TPA directly activates pig adrenal steroidogenesis, resulting in increased Cortisol protein kinase C. Vallotton, Capponi, Grillet, Knupfer, Hepp, production. Khosla and Bumpus (1981) have shown that angiotensin II stimulates Cortisol production in bovine fasciculata cells by a Fig. 1 illustrates the profile of protein kinase C activity observed when guinea pig adrenocortical tissue cytosol was analyzed by DEAE-cellulose chromatography. A distinct peak of Ca/phospholipid-dependent kinase eluted at 0.06 M NaCl. Peak of type II cAMP-dependent protein kinase which was eluted at 0.18 M NaCl was separated from protein kinase C, as shown in Fig. 1.

Downloaded by: NYU. Copyrighted material.

Role ofProtein Kinase Cin Cortisol Production

Horm. metabol. Res. 22 (1990)

T. Nishikawa, Akiko Yoshida, Y. TamuraandS.

Yoshida

Roskoski R. Jr.:Assays of protein kinase. Methods in Enzymology 99: 3-6(1983) Simpson, E. R.: Cholesterol side-chain cleavage, cytochrome P450, and the control of steroidogenesis. Mol. Cell. Endocr. 13:213-227 (1979) Takai, Y., A. Kishimoto, M. Inoue, Y. Nishizuka: Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum. J. Biol. Chem. 252: 7603-7609 (1977) Uzquiris, V. L., C. A. Whipple, H. A. Salhanick:Steroselective inhibition of cholesterol side-chain cleavage by enantiomers of aminoReferences glutethimide. Endocrinology 101: 89-92 (1977) Vallotton, M.B..A.M. Capponi, C. Grillet, A. L. Knupfer, R. Hepp, M. Brami, B., I. Vilgrain, E. M. Chambaz: Sensitization of adrenocortical C. Khosla, F. M. Bumpus: Characterization of angiotensin recepcell adenylate cyclase activity to ACTH by angiotensin II and actitors on bovine adrenal fasciculata cells. Proc. Natl. Acad. Sci. vators of protein kinase C. Mol. Cell. Endocrinol. 50: 131-137 USA. 78:592-596 (1981) (1987) Vilgrain, I., C. Cochet, E. M. Chambaz: Hormonal regulation of a calBrunswig, B., A. K. Mukhopadhyay, L. T. Budnik, H. G. Bohnet, A. cium-activated, phospholipid-dependent protein kinase in bovine Leidenberger: Phorbol ester stimulates progesterone production adrenal cortex. J. Biol. Chem. 259:3403-3406 (1984) by isolated bovine luteal cells. Endocrinology 118: 743-749 Vilgrain, I., G. Defaye, E. M. Chambaz: Adrenocortical cytochrome P(1986) 450 responsible for cholesterol side chain clevage (P-450scc) is Castagna, M., Y. Takai, K. Kaibuchi, K. Sano, U. Kikkawa, Y. Nish- phosphorylated by the calcium-activated, phospholipid-sensitive izuka: Direct activation of calcium-activated, phospholipid-deprotein kinase (protein kinase C). Biochem. Biophys. Res. Compendent protein kinase by tumor promoting phorbol esters. J. Biol. mun. 125:544-561 (1984) Chem. 257:7847-7851 (1982) Culty, M., I. Vilgrain, E. M. Chambaz: Steroidogenic properties of phorbol ester and a Ca + 2 ionophore in bovine adrenocortical cell Requests for reprints should be addressed to: suspensions. Biochem. Biophys. Res. Commun. 121: 499-506 (1984) Finn, F. M., C. Stehle, P. Ricci, K. Hofmann: Angiotensin stimulation Tetsuo Nishikawa, M. D., Ph. D. of adrenal fasciculata cells. Arch. Biochem. Biophy. 264:160-167 Department of Internal Medicine (II) (1988) School of Medicine Hadjian, A. J., C. Guidicelli, E. M. Chambaz:Cholinergic muscarinic Chiba University stimulation of steroidogenesis in bovine adrenal cortex fasciculata 1-8-1 Inohana, Chiba City cell suspensions. Bioch. Biophy. Acta714:157-163 (1982) Chiba 280 (Japan) Hadjian, A. J., M. Culty, E. M. Chambaz: Stimulation of phosphatidylinositol turnover by acetylcholine, angiotensin II and ACTH in bovine adrenal fasciculata cells. Bioch. Biophy. Acta 804:427-433 (1984a) Hadjian, A. J., M. Culty, E. M. Chambaz: Rapid polyphosphoinositide decrease is an early event in the steroidogenic response of bovine adrenocortical fasciculata cells to angiotensin II. Biochem. Biophy. Res. Commun. 124:393-399 (1984b) Halkerston, I. D. K.: Cyclic AMP and adrenocortical function. Adv. Cycl. Nucl. Res. 6:99-136 (1975) Honma, M., T. Satoh, J. Takezawa, M. Ui: An ultrasensitive method for the simultaneous determination of cyclic AMP and cyclic GMP in small volume samples from blood and tissues. Biochem. Med. 18:257-273(1977) Kenyon, C. J., L. Anyaorah, L. Woodburn, J. M. C. Connell, R. Eraser: Stimulation of Cortisol production in isolated bovine zona fasciculata cells by phorbol ester: role of ion fluxes. J. Endocr. 117:423429(1988) Kikkawa, U., R. Minakuchi, Y. Takai, Y. Nishizuka: Calcium-activated, phospholipid-dependent protein kinase (protein kinase C) from rat brain. Methods in Enzymology 99:288-298 (1983) Kojima, I., K. Kojima, D. Kreutter, H. Rasmussen:The temporal integration of the aldosterone secretory response to angiotensin occurs via two intracellular pathways. J. Biol. Chem. 259: 14448-14457 (1984) Lowry, O. H., N. J. Rosebrough, A. L. Fair, R. J. Randall: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275(1951) Minakuchi, R., Y. Takai, B. Yu, Y. Nishizuka: Widespread occurrence of calcium-activated, phospholipid-dependent protein kinase in mammalian tissues. J. Biochem. 89:1651-1654(1981) Nishikawa, T., C. A. Strott:Cortisol production by cells isolated from the outer and inner zones of the adrenal cortex of the guinea pig. Endocrinology 114:486-491 (1984) Podesta, E. J., A. Milani, H. Steffan, R. JVe/ier:Steroidogenic action of calcium ions in isolated adrenocortical cells. Biochem. J. 186: 391-397(1980)

In conclusion, we have demonstrated the existence of Ca/phospholipid-dependent protein kinase in guinea pig adrenals. The action of TPA appears not to require the generation of cAMP, and the site of action of TPA is located at a point before the production of pregnenolone in mitochondria. These results indicate that protein kinase C may be involved in modulating guinea pig adrenocortical cell steroidogenesis in addition to the classical cAMP-dependent protein kinase pathway.

Downloaded by: NYU. Copyrighted material.

32

Involvement of protein kinase C in the regulation of cortisol production by guinea pig adrenocortical cells.

Cytosol of the guinea pig adrenals was found to contain a protein kinase which was dependent on the presence of both calcium and phospholipids (phosph...
403KB Sizes 0 Downloads 0 Views