Specific Regulation by Steroid Hormones of Protein Kinases in the Endometrium

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1 Eur. J. Biochem. 104, (1980) Specific Regulation by Steroid Hormones of Protein Kinases in the Endometrium 2. Alteration in Levels of Protein Kinases in Human Endometrium during the Menstrual Cycle Kohji MIYAZAKI, Eishichi MIYAMOTO, Masao MAEYAMA, and Makio UCHIDA Department of Obstetrics and Gynecology, Pharmacology, and Biochemistry, Kumamoto University Medical School (Received August 6, 1979) The alteration in the levels of multiple protein kinases has been studied in human endometrium during the menstrual cycle. Protein kinases were examined in the cytosol fraction and separated by DEAE-cellulose column chromatography. The level of type I adenosine 3,5 -monophosphatedependent (CAMP-dependent) protein kinase was higher than that of type I1 CAMP-dependent protein kinase at the proliferative phase. At the late secretory phase, type I CAMP-dependent protein kinase markedly decreased, whereas type I1 CAMP-dependent protein kinase remained at the same level. Therefore, type I1 CAMP-dependent protein kinase was exclusively predominant at this phase. CAMP-dependent and CAMP-independent protein kinases with protamine increased more at the late secretory phase than at the proliferative phase. The results presented demonstrate a specific alteration in protein kinase activities during the menstrual cycle and are compatible with those obtained in rabbit endometrium (the preceding paper). The preceding paper [l] has reported the specific regulation by steroid hormones of multiple protein kinases in rabbit endometrium. The levels of types I and I1 CAMP-dependent protein kinases have been separately controlled by treatment with estrogen and progesterone. Furthermore, CAMP-dependent protein kinase with protamine and CAMP-independent protein kinases with protamine, casein and phosvitin have also been regulated by the hormones. These results suggest that the levels of protein kinases in the endometrium, a target organ for the steroid hormones, change under regulation of specific hormones during the menstrual cycle. The present paper describes the alteration in the levels of multiple protein kinases in human endometrium during the menstrual cycle. The uteri were obtained by medically unavoidable hysterectomy. Evidence will be presented that protein kinases in the tissue are regulated in the natural state under influence of specific hormones during the menstrual cycle. Abbreviations. CAMP, adenosine 3,5 -monophosphate; EGTA, [ethylene bis(oxoethylenenitrilo)]tetraacetic acid. Enzyme. Protein kinase or ATP: protein phosphotransferase (EC ). MATERIALS AND METHODS Collection of Samples Endometrial tissue samples were obtained from the uteri of 15 female subjects who underwent medically warranted abdominal hysterectomy. All of the patients had a history of normal regular menstrual cycles, their cycle length being days, and had no endocrine disorder. Their ages varied from 39 to 46 years with an average of 43 years. Thirteen of them suffered from myoma of the uterus and two of the rest from carcinoma of the cervix uteri (carcinoma in situ). None of them received hormonal therapy and medicine to induce hormone secretion. The endometrium was macroscopically separated from the myometrium with scissors. Immediately after collection, an endometrial specimen was partly saved for histological examination. The rest of the tissue was placed in ice-cold physiological saline and rinsed to remove the blood. The tissue obtained was confirmed to have no abnormal lesion macroscopically and microscopically. The samples were divided into three groups according to the histological dating of the endometrium [2].These groups corresponded to the proliferative (1st- 15th day), early secretory

2 544 Specific Regulation by Steroid Hormones of Protein Kinases. 2 (16th-23rd day) and late secretory phases (24th- 28th day). No selection of samples was made except for the above conditions of patients. The average weight of tissue obtained was 1.3 g. The range varied from 0.2 to 3.6 g. The number of the tissue samples used was five for each phase. Each assay was made for the respective samples in duplicate to quadruplicate determinations. The mean obtained from each sample was used for further calculation. Preparation of Cytosol Fraction All procedures were carried out at 0-4 C. After weighed, the tissue was homogenized with 5 vol. of 0.25 M sucrose and 10 mm Tris-C1 buffer ph 7.5, at 900 rev./min in a glass homogenizer with Teflon pestle of 0.15-mm clearance. The homogenate was centrifuged at x g for 60 min. The resultant clear supernatant solution, designated cytosol fraction, was used for further experimentation. The other materials and methods used in the present study were essentially as described previously [l]. RESULTS Protein Kinase Activities in the Cytosol Fraction during the Menstrual Cycle The alteration in protein kinase activities in the cytosol fraction during the menstrual cycle was examined using several substrates such as histone, protamine, casein and phosvitin. The activities were analyzed from three respects, i.e. activity per g wet tissue, activity per mg protein (specific activity) and activity per mg DNA. In general, the alteration in protein kinase activities in the cytosol fraction with respective substrates was not great during the phases of the menstrual cycle. The activity per mg protein with histone decreased at the shift from the early secretory phase to the late secretory phase. The activity per g wet tissue with casein increased about twice at the shift from the proliferative phase to the early secretory phase and then decreased to the original level at the late secretory phase. The mean values of the ratio of protein kinase activities determined in the absence and presence of CAMP were 0.27, 0.36 and 0.34 at the proliferative, early secretory and late secretory phases, respectively. From the repeated experiments, the ratio of activity were similar to each other and considered not to be significant during the menstrual cycle. Activity Profile of CAMP-Dependent and CAMP-Independent Protein Kinases during the Menstrual Cycle on DEAE-cellulose Column The alteration in protein kinase activities in the cytosol fraction was not great on the basis of any parameter during the menstrual cycle. However, protein kinases in the cytosol fraction of the tissue are in multiple forms, each of which may differently alter during the menstrual cycle. Therefore, the activity profile on DEAE-cellulose column was examined with several substrates including histone, protamine, casein and phosvitin. For the convenience of comparison, the cytosol fraction containing similar amount of protein was applied to the same size of the column. The typical results with histone as substrate are shown in Fig. 1. Both types I and I1 CAMP-dependent protein kinases are present in the tissue at the proliferative phase (Fig.1A). The peak of type I CAMPdependent protein kinase is higher than that of type I1 CAMP-dependent protein kinase. When the phase shifts from proliferative to early secretory, both types I and I1 CAMP-dependent protein kinases remained at the similar level (Fig. 1 A and B). The peaks of CAMP-binding activity were coincident with those of enzyme activity. A peak of CAMP-binding activity which did not coincide with activity peak was eluted with about 0.12 M NaCl (Fig. 1 B). At the late secretory phase, the peak of type I CAMP-dependent protein kinase markedly decreased, whereas the peak of type I1 CAMP-dependent protein kinase remained at the same level in comparison to that at the early secretory phase. Thus, type I1 CAMP-dependent protein kinase was exclusively predominant at this phase. At the late secretory phase, the peak of CAMP-binding activity eluted with 0.12 M NaCl was prominent (Fig. 1 C). When protamine was used as substrate, the activity profile on DEAE-cellulose column is shown in Fig. 2. Two active peaks were eluted with about 0.08 and 0.18 M NaCl. The first peak of activity (peak 1) was not stimulated with CAMP, while the second peak (peak 2) was stimulated with CAMP. Both peaks 1 and 2 increased at the late secretory phase (see Fig. 2A and C). The results obtained with histone (Fig. l), protamine (Fig.2), phosvitin and casein as substrate are summarized in Table 1. The protein kinase activity of each peak with respective substrates was integrated by the profile on DEAE-cellulose column. The validity of the data was confirmed by statistical examination. The activity ratio of type I1 CAMP-dependent protein kinase to type I CAMP-dependent protein kinase did not markedly change at the shift from the proliferative to the early secretory phase. At the late sectory phase, the peak of type I CAMP-dependent protein kinase decreased, while that of type I1 CAMP-dependent protein kinase remained at the similar level. Thus, the activity ratio of type I1 to type I increased to 8.8. The levels of both peaks 1 and 2 with protamine as substrate increased at the late secretory phase. The activity ratio of peak 2 to peak 1 did not change. The

3 K. Miyazaki, E. Miyamoto, M. Maeyama, and M. Uchida ( A Proliferative phase / 10.4 t n /' r B + Early secretory phase 1 15 [ A Proliferative phase 10 2o r A Early secretory phase C Late secretory phase 15 - ka /- 60 / Fraction number Fig. 1. Activity profile with histone on DEAE-cellulose column of the cytosol fraction during the menstrual cycle. The cytosol fraction containing 16.3 mg protein in 2.1 ml, 15.8 mg protein in 3.5 ml and 17.3 mg protein in 3.5 ml for the proliferative phase (A), the early secretory phase (B) and the late secretory phase (C), respectively, was applied to a 1.1 x 5.3-cm column of DEAE-cellulose. The elution procedures of enzyme protein were as described previously [I]. The aliquots (0.075 ml) of the fractions were assayed for protein kinase activity with histone as substrate in the presence (M) or absence (G-4) of 1 pm CAMP. The other aliquots (0.08 ml) were assayed for CAMP-binding activity (A---A) - 20 c 9 Late secretory phase ), Fraction number Fig. 2. Activity profile with protarnine on DEAE-cellulose column of the cytosol fraction during the menstrual cycle. The cytosol fraction containing 16.3,15.8 and 17.3 mg protein for the proliferative phase (A), the early secretory phase (B) and the late secretory phase (C), respectively, was applied to a 1.1 x 5.3-cm column of DEAE-cellulose. Each of the experiments (A-C) represented corresponds to that shown in Fig. 1. The elution procedures of enzyme proteins were as described previously [I]. The aliquots (0.075 ml) of the fractions were assayed for protein kinase activity with protarnine as substrate in the presence (t-w) or absence (b--a) of 1 pm CAMP 13 Q Table 1. Analysis of the peak of activity per mg protein on DEAE-cellulose column Integrated protein kinase activity per mg protein in the cytosol fraction was calculated for the peak of activity with each substrate on DEAEcellulose column. The activity of' each peak with respective substrates at the proliferative phase was taken as 100 per cent and from this value, the activity of peak for each phase was calculated as percentage. Parentheses represent the number of the experiments Phase of menstrual cycle Protein kinase activity per mg protein Histone Protamine Phosvitin Casein Type 1 Type II Type I1 Peak 1 Peak 2 Peak 2 % Peak 1 Proliferative (5) " 1OOk11 2.4f0.4b " loo+ 8" f Early secretory (5) 121 f 15b 115 k f 0.4b 121 k f f f 21 Late secretory (5) "s' 120 f f 1.4,c 173 i 26" 148 & 10" 1.6 f & P < P < P <

4 546 Specific Regulation by Steroid Hormones of Protein Kinases. 2 Table 2. The apparent K, values for CAMP of types I and II CAMPdependent protein kinases at each phase during the menstrual cycle The respective enzyme preparations were separated on DEAEcellulose column. The collected fractions were concentrated by Amicon ultrafiltration with PM-10 membrane and dialyzed against 10 mm Tris-C1 buffer ph 7.5, 1 mm MgZ+ and 0.05 mm EGTA. The enzyme protein used was 34.1, 37.5,29.3, 11.7, 17.4 and 16.5 pg of types I and I1 protein kinases for the proliferative, early secretory and late secretory phases, respectively. The other experimental conditions were as described in Materials and Methods Phase of menstrual cycle K, value type I M type I1 Proliferative 2.5 x x 10-9 Early secretory 1.3 x x 10-9 Late secretory 5.9 x x 10-9 peaks of activity with phosvitin and casein did not greatly change at each phase. K, Values for camp of Types I and II CAMP-Dependent Protein Kinases at Each Phase of the Menstrual Cycle In order to examine how the affinity of types 1 and I1 CAMP-dependent protein kinases for camp changes during the menstrual cycle, the apparent K, values for camp were determined at each phase (Table 2). The values are in the range of 1.3 to 5.9 x M. The K,,, values of types I and 11 CAMPdependent protein kinases were lowest at the early secretory phase. Although the level of type I CAMPdependent protein kinase markedly decreased and the level of type I1 CAMP-dependent protein kinase remained at the same level at the shift from the proliferative to the late secretory phase (see Fig.l), the K, values of both enzymes only slightly increased. It appeared unlikely that correlation occurs between the change of enzyme activity and that of K, for CAMP. DISCUSSION The experimental data obtained in the present study are compatible with the supposition that the levels of multiple protein kinases in the cytosol fraction of the endometrium alter under influence of specific hormones during the menstrual cycle. The level of type I CAMP-dependent protein kinase is high at the proliferative phase (see Fig. 1). This agrees well with the phenomenon in which type I CAMPdependent protein kinase increases by treatment with estrogen [I]. Estrogen secreted from the follicle in the ovary is known to affect the function of the endometrium during the proliferative phase of the men- strual cycle [3,4]. Thus, evidence has been presented that type I CAMP-dependent protein kinase is regulated by estrogen in relation with proliferation of cells. On the other hand, type I1 CAMP-dependent protein kinase is exclusively predominant at the late secretory phase (see Fig.1C). This is in accordance with the results that type I1 CAMP-dependent protein kinase increased by treatment with progesterone [l] and that the activity profile of human endometrial enzymes on DEAE-cellulose column at the late secretory phase (see Fig. 1 C) closely resembled that of rabbit endometrium after ovulation caused by treatment with human chorionic gonadotropin [l]. At the secretory phase of the menstrual cycle, progesterone secreted from the corpus luteum in the ovary after ovulation mainly regulates the function of the endometrium [3,4]. The data indicate that type I1 CAMP-dependent protein kinase is primarily controlled by progesterone in association with differentiation of cells. In addition, the results presented also suggest that types I and I1 CAMP-dependent protein kinases may have separate roles in the endometrium. The alteration in levels of protein kinases, as was found, may be based on de novo synthesis of the enzyme protein. (a) The menstrual cycle comes round in several days under anabolic effects of specific hormones. The relatively long period of chronic treatment was required to produce the effect of estrogen or progesterone on protein kinase levels in rabbit endometrium [l]. (b) The increase of protein kinases was associated with the increase of tissue weight and amounts of protein and DNA [l]. (c) The simultaneous treatment with cycloheximide, an inhibitor at translational stage for protein synthesis, abolished the specific effect of estrogen or progesterone 111. (d) It appeared unlikely that the alteration in protein kinase activities in human and rabbit endometria is due to that of sensitivity of the enzymes for CAMP. In view of substrate specificity, independence of camp and elution position on DEAE-cellulose column, a peak of activity with protamine (peak l), eluted with about 0.08 M NaCl, may be identical with a proenzyme from brain tissue, which is convertible to an active enzyme by partial proteolysis [5,6]. This protamine kinase is different from type I CAMP-dependent protein kinase, although both enzymes are eluted at the similar position on the DEAEcellulose column. It was shown in the present investigation that estrogen regulates preferentially type I CAMP-dependent protein kinase, whereas the protamine kinase is regulated under influence of progesterone, confirming that these enzymes are separate entities. It remains to be elucidated whether the mechanism of the alteration in protein kinase levels is physiologically significant since the roles of protein kinases

5 ~~ K. Miyazaki, E. Miyamoto, M. Maeyama, and M. Uchida 547 in the endometrium are unknown. On the other hand, a variety of evidence suggests involvement of cyclic nucleotides and their related substances in the function of the target organs for steroid hormones. Several agents such as estrogen and prostaglandins which are known to affect the uterus increase levels of camp [7-91 and cgmp [10,11] in the uterus and oviduct. Contradictorily, some investigators have reported that estrogen slightly decreases the level of camp [lo,ll]. camp content altered in the endosalpinx of the oviduct during the menstrual cycle [12]. The administration of exogenous camp mimics the estrogen-like anabolic effect [13] and estrogen-induced stimulation of glycolytic enzymes [14]. The simultaneous administration of theophylline enhanced the effect of camp [14]. Steroid hormones caused the alteration in levels of protein kinases [ and regulation of phosphorylation of specific protein present in the cytosol fraction from target organs [18]. In addition, it has been reported that the cytoplasmic estradiol receptor in the endometrium can be phosphorylated by CAMP-dependent protein kinase from the myometrium and that this, in turn, results in an increase of nuclear RNA polymerase activity [19]. The occurrence of CAMP-dependent protein kinase has been reported in the endometrium of the bovine [20], mouse [15], rabbit [l] and human (the present study). There is a possibility that CAMP-dependent protein kinases may play a physiological role by phosphorylating the natural substrate or substrates in the endometrium. The mechanism of the action may be based on the activation of CAMP-dependent protein kinases by CAMP [21,22]. REFERENCES 1. Miyazaki, K., Miyamoto, E., Maeyama, M. & Uchida, M. (1980) Eur. J. Biochem. 104, , 2. Noyes, R. W., Hertig, A. T. & Rock, J. (1950) Fertil. Steril. I, Wiele, R. L. V., Bogumil, J., Dyrenfurth, I., Ferin, M., Jewelewiez, R., Warren, M., Rizkallah, T. & Mikhail, G. (1970) Recent Progr. Horm. Res. 26, Hillard, J. & Eaton, L. W., Jr (1971) Endocrinology, 89, Takai, Y., Kishimoto, A., Inoue, M. & Nishizuka, Y. (1977) J. Biol. Chem. 252, Inoue, M., Kishimoto, A., Takai, Y. & Nishizuka, Y. (1977) J. Bid. Chem. 252, Rosenfeld, M. G. & O Malley, B. W. (1970) Science (Wash. DC), 168, Szego, C. M. & Davis, J. S. (1967) Proc. Natl Acad. Sci. U.S.A. 58, Mala, H., Jr, Deal, M., Hodgson, B. & Pauerstein, C. J. (1975) Fertil. Steril. 26, Kuehl, F. A,, Jr, Ham, E. A,, Zanetti, M. E., Sanford, C. H., Nicol, S. E. & Goldberg, N. D. (1974) Proc. Natl Acad. Sci. 1J.S.A. 71, Flandroy, L. & Galand, P. (1978) J. Cyclic Nucleotide Res. 4, Munemura, M., Tazoe, Y., Ozaki, H., Nakahara, K. & Maeyama, M. (1979) Fertil. Steril. 31, Hechter, O., Yoshinaga, K., Halkerston, I. D. K. & Birchall, K. (1967) Arch. Biochem. Biophys. 122, Singhal, R. L. & Lafreniere, R. T. (1972) J. Pharmacol. Exp. Ther. 180, D$skeland, S. O., Kvinnsland, S. & Ueland, P. M. (1975) J. Reprod. Fertil. 44, Lee, P. C., Radloff, D., Schweppe, J. S. & Jungmann, R. A. (1976) J. Biol. Chem. 251, Fuller, D. J. M., Byus, C. V. & Russell, D. H. (1978) Proc. Natl Acad. Sci. U.S.A. 75, Liu, A. Y.-C. & Greengard, P. (1976) Proc. Natl Acad. Sci. U.S.A. 73, Arnaud, M., Beziat, Y., Borgna, J. L., Guilleux, J. C. & Mousseron-Canet, M. (1971) Biochim. Biophys. Acta, 254, Sanborn, B. M., Bhalla, R. C. & Korenman, S. G. (1973) J. Biol. Chem. 248, Kuo, J. F. & Greengard, P. (1969) Proc. Natl Acad. Sci. U.S.A. 64, Greengard, P. & Kuo, J. F. (1970) in Role of Cyclic AMP in Cell Function (Greengard, P. and Costa, E., eds) pp , Raven Press, New York. K. Miyazaki and M. Maeyama, Department of Obstetrics and Gynecology, Kumamoto University Medical School, Honjo, Kumamoto-shi, Kumamoto-ken, Japan 860 E. Miyamoto*, Department of Pharmacology, Kumamoto University Medical School, Honjo, Kumamoto-shi, Kumamoto-ken, Japan 860 M. Uchida, Department of Biochemistry, Kumamoto University Medical School, Kumamoto-shi, Kumamoto-ken, Japan 860 * To whom correspondence should be addressed.