Download pdf - Hormonal regulation of PKC-δ protein and mRNA levels in the rabbit corpus luteum

Molecular and Cellular Endocrinology 122 (1996) 213-221

MO’ ocular and

Celluisr

-

Hormonal regulation of PKC-6 protein and mRNA levels in the rabbit corpus luteuml

Evelyn T. Maizels, Malathy Shanmugam, Marilyn L.G. Lamm, Mary Hunzicker-Dunn*

Department of Cell and Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA

Received 3 January 1996; accepted 13 July 1996

Abstract

We have previously reported that rabbit corpora lutea exhibit a prominent phosphorylated substrate protein at 76 kDa which corresponds to the autophosphorylated form of protein kinase C (PKC) 6 and that the expression of PKC-6 protein is increased in rabbit corpora lutea of pseudopregnancy at least 2-fold when serum estrogen levels are raised by the presence of an estrogen implant inserted at the time of human chorionic gonadotropin (hCG)-induced ovulation. The purpose of the experiments described herein was to evaluate further the hormonal regulation of PKC-6 in the rabbit corpus luteum. Results demonstrate that luteal PKC-6 protein and mRNA are concomitantly induced some S-fold within 48 h in response to an ovulatory surge of hCG; that, as in corpora lutea of pseudopregnancy, luteal PKC-6 expression is relatively constant during the life span of the corpus luteum following a fertile mating; that exogenous estrogen does not modulate the induction of luteal PKC-6 during luteinization but promotes an additional two-fold increase in steady state PKC-8 mRNA (and protein) levels in corpora lutea by day 10 of pseudopregnancy; and that luteal PKCd expression can be abruptly and reversibly modulated upon withdrawal and subsequent replacement of an estrogen implant to pseudopregnant rabbits. These results demonstrate that an ovulatory surge of luteinizing hormone induces the expression of PKCd mRNA and protein in rabbit corpora lutea, and that once the corpus luteum becomes estrogen responsive, estrogen then regulates expression of PKC-6 mRNA and protein.

Keywords: Protein kinase C; Luteinizing hormone; Estrogen; Corpus luteum

1. Introduction

Maintenance of pregnancy in the rabbit requires progesterone synthesized by the corpus luteum. While formation of the corpus luteum in the rabbit, as in all other species, is prompted by the preovulatory surge of LH which leads to ovulation and luteinization of ovarian follicles, luteal function after day 5 is dependent upon estrogen supplied to the corpus luteum by nearby ovarian follicles (for review see [l]). The devel-

*Corresponding author. Tel: + 312 503 8940; fax: + 312 503 0566; email: [email protected]

’ This work was supported by NIH Grants HD-28472 (to M.H.D.)

and the Northwestern University Center for Reproductive Science (P30-HD-28048).

opment of the dependence of the corpus luteum on estrogen [2,3] coincides with the appearance of estrogen receptors in the corpus luteum between days 4 to 6 of pseudopregnancy [4,5]; luteal estrogen receptors then persist until luteal demise [5-71. Estrogen alone will maintain the corpus luteum in a fully functional state in the absence of the pituitary [8]. However, as elevation of serum estrogen above physiological levels does not modify serum progesterone levels [9-121, one of the most useful models to evaluate the dependence of rabbit luteal functions on estrogen is the one developed by Holt et al. [lo]. In this model, corpora lutea become dependent upon an exogenous source of estrogen, supplied from an implant containing crystalline estradiol- 17p inserted at the time of the induction of pseudopregnancy. Removal of the implant on days 9 to 10 of pseudopregnancy results in a rapid decline in

0303-7207/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved

PII SO303-7207(96)03885-3

serum progesterone levels; replacement of the implant during the ensuing 48 h rescues corpus luteum function, including progesterone biosynthesis [lo, 13- 171. Utiliz- ing this model, investigators have demonstrated that estrogen does not modulate the catalytic activity of the enzymes of the steroidogenic pathway, including 3-hydroxy-3-methylglutaryl-coenzyme A reductase, cholesterol esterase, cholesterol acetyltransferase, or P-450 cholesterol side chain cleavage (P-450,sso) [ 15,17,18]. Rather, the site of estrogen’s action in progesterone biosynthesis has been localized to points downstream of cholesterol esterase and upstream of the association of cholesterol with the catalytic site on P-450,,,, [ 18,191.

We have recently demonstrated that insertion of an estrogen implant at the time of the induction of pseudopregnancy selectively up-regulates the content of the protein kinase C [PKC] isoform PKC-6 in rabbit corpora lutea [20]. PKCs comprise a ubiquitously dis- tributed family of serine/threonine protein kinases which are involved in such diverse cellular processes as metabolism. membrane transport. and secretion as well

as the regulation of gene expression leading to changes in cellular differentiation and proliferation [2 1.221. Cur- rently, twelve different isotypes have been identified [21]. These different isotypes are classified based upon their biochemical properties and amino acid sequences: the ‘conventional’ isotypes require Ca’ + , phospholipids and diolein for activation and include c(, p, and y; the ‘novel’ isotypes require only phospholipids and diolein for activation and include 6, E, rl. 8, u; the ‘atypical’ isotypes require only phospholipids for activation and include c, h, and r [21,22]. However, neither the hormonal regulation of the expression nor the function of specific PKC isotypes is well-understood [21,22].

We have identified PKC-6 as a prominent phosphorylation activity in soluble rabbit luteal extracts [20]. Exogenous estrogen, introduced as an estrogen implant at the time of the preovulatory hCG injection to induce pseudopregnancy, increased PKC-F protein 2-fold in corpora lutea on days 5. 10 and 15 of pseudopregnancy [20]. We have detected the increase in luteal PKC-S protein expression not only as an immunoreactive band at 76 kDa but also by its autophosphorylation under our standard phosphorylation conditions which activate ‘novel’ isotypes of PKC (i.e. in the presence of diolein and phospholipids), as well as under acid phosphorylation conditions [23] which promote cofactor-independent activation of PKCs [20]. In contrast to

PKC-S, estrogen did not regulate the expression of PKC-cx or -p in rabbit corpora lutea [20].

The purpose of the experiments described herein was to evaluate further the hormonal regulation of PKC-8 in the rabbit corpus luteum. Specifically, we sought to determine when during luteal formation was PKC-S induced. if estrogen regulated not only PKC-6 protein but also steady state mRNA levels, if PKC-S expression

was regulated during pregnancy in corpora lutea, and if continued expression of PKC-F in corpora lutea of pseudopregnancy was dependent on estrogen as as- sessed using the rabbit luteal model developed by Holt et al. [lo]. Results demonstrate that PKC-6 protein and mRNA are concomitantly induced with luteinization in response to the ovulatory surge of gonadotropin, that exogenous estrogen increases not only PKC-6 protein but also steady state mRNA levels an additional 2-fold in corpora lutea during pseudopregnancy, that PKC-6 expression is relatively constant during the life span of the corpus luteum following a fertile mating, and that like progesterone production, luteal PKC-S expression can be abruptly and reversibly modulated upon withdrawal and subsequent replacement of estrogen implants to pseudopregnant rabbits.

2. Materials and methods

2.1. Muteriuls

The following materials were purchased: [Y-~‘P]ATP (ammonium salt; SA, 3000 Ci/mmol) and [c1- “P]deoxyCTP (ammonium salt; 3000 Ci/mmol) from DuPont-New England Nuclear (Boston, MA); SDS- PAGE reagents from Bio-Rad (Richmond, CA); TRI- zol from Gibco-BRL (Grand Island, NY); and enhanced chemiluminescence (ECL) reagents and Hy- bond-C membranes from Amersham Life Science, Inc (Arlington Hts., IL). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

2.2. Animals and tissue preparation

Follicles or corpora lutea were dissected from ma- ture, often retired, female breeder rabbits (primarily obtained from Keiper Rabbit Farm, Gary, IN). Pseu- dopregnancy was induced on day 0 by the intravenous injection of 100 IU hCG (Ayerst Laboratories, NY) in 0.9% (w/v) NaCl. Pregnancy was induced by placing fertile males with females. The day after hCG injection or mating was counted as day 1 of pseudopregnancy or pregnancy, respectively. Estrogen-treated rabbits received 1 .O-cm silastic implants [lo] containing estradiol- 178 on day 0 of pseudopregnancy, unless otherwise indicated. These implants yielded a 6-fold increase in serum estrogen levels in day-10 pseudopregnant rabbits compared to unimplanted controls [20]. Animal care and treatments were in accordance with the NIH and USDA Guidelines and were approved by the Animal Care and Use Committee of Northwestern University.

Soluble extracts of follicles and corpora lutea were prepared by homogenization in 36 volumes in a buffer containing 10 mM Tris-HCl, pH 7.5, 4 mM MgCl,, 1 .O mM EGTA, 0.32 M sucrose with 15 strokes in a

E.T. Maizels et al. / Molecular and Cellular Endocrinology 122 (1996) 213-221 215

ground glass homogenizer. The homogenate was cen- trifuged at 105 000 x g for 70 min at 4°C. Protein concentrations in the soluble fraction were measured by the method of Lowry et al. 1241 using crystalline BSA as a standard.

2.3. Phosphorylation reaction

Phosphorylation of PKC-S was conducted, as previously described [25,26] in an incubation volume of 220 pl, containing 100 l.11 of soluble extract ( w 100 l.tg protein) and 100 ~1 reaction buffer (10 pmol of a-glyc- erol phosphate buffer, pH 7.0, 100 nmol of dithiothrei- tol, 2 pmol of MgCl,, and 1 nmol ATP). Preincubation was performed for 3 min at 37°C; the phosphorylation reaction was initiated by the addition of 5 uCi of [y-32P]ATP/0.22 ml reaction volume (yielding a final specific activity of _ 1 x lo6 cpm/pmol) in the presence of 45 ug/ml phosphatidylserine, 1.6 pg/ml 1,Zdiolein and 0.5 mM EGTA. After incubation for 1 min at 37°C the reaction was terminated with the addition of 0.5 vol SDS stop solution [25] followed by heat denatu- ration (1 OO”C, 5 min). 32P-labeled cellular proteins were separated by SDS-PAGE [25] using 5% (w/v) stacking and 8.5% (w/v) separating gels. Molecular weight stan- dards and processing of gels for autoradiography is as previously described [20,25]. Quantitation of [32P]phosphate incorporation was determined by densit- ometric scanning of autoradiograms, using a Zeineh soft laser densitometer with Hewlett Packard 3390 A integrator or on Fujix bioimaging analyzer BAS 2000, following exposure of dried gels to Fuji imaging plate type BAS III.

2.4. Western blot analysis

Proteins (120 pg) in soluble extracts were separated by SDS-PAGE and transferred to Hybond-C nitrocel- lulose membranes. PKC-8 was detected by ECL using an amino thermal peptide-directed antibody purchased from Transduction Laboratories (Lexington, KY).

2.5. RNA blot analysis.

Tissues were dissected and immediately frozen in liquid nitrogen. Total RNA was prepared using TRIzol reagent, following manufacturer’s instructions. Ten pg of total RNA was separated by electrophoresis on a 1 % (w/v) formaldehyde-agarose gel [27], transferred to a nylon membrane and covalently cross-linked with a UV stratalinker, and hybridized to a 2-kilobase PKC-6 cDNA that had been labeled with [ol-32P]deoxyCTP using random hexamer primers and the Knenow fragment of Escherichia coli DNA polymerase [27]. RNA blots were re-probed with a CHO-B probe that detects the LLrep3 gene family to control for RNA loading.

Hybridizations were carried out in 50% (w/v) for- mamide, 5 x SSP (0.9 % NaCl, 50 mM sodium phosphate, pH 7.7, 5 mM EDTA), 2 x Denhardt’s reagent, 10 % dextran sulfate, 0. 1 % (w/v) SDS, and 100 pg/ml salmon sperm DNA at 42°C. Membranes were washed in 1 x SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0) at 50°C and exposed to Kodak XAR-5 film. Quan- titations were as described above for [32P]-labeled proteins.

2.6. RIA

Blood was obtained immediately following animal euthanasia by heart puncture. Progesterone levels were determined in serum samples obtained on the indicated days of pseudopregnancy using a kit from ICN Bio- chemicals, employing [1251]progesterone as the tracer by the Northwestern University Center for Reproductive Science Hormone and Neurotransmitter Core. The in- ter- and intraassay coefficients of variance were 16% and 8”/0, respectively.

2.7. Statistical analyses

Effects of treatments were evaluated by the unpaired Student’s t-test. When indicated, effects of treatments were also evaluated by analysis of variance and Dun- can’s multiple range test.

3. Results

Based upon preliminary studies demonstrating that PKC-S expression was relatively low in rabbit ovarian preovulatory follicles [Maizels and Hunzicker-Dunn, unpublished data] and markedly elevated by day 5 of pseudopregnancy [20], studies were conducted to ascer- tain when PKC-S expression was increased during luteal development. We have previously established that [32P]PKC-6, phosphorylated during an autophosphorylation reaction performed under in vitro conditions which selectively activate the ‘novel’ isotypes of PKC, accurately reflects the protein concentration of this PKC determined by Western blot analysis [20]. We have selected to utilize primarily the autophosphorylation of PKC-6 to assess PKC-6 protein content due to the high sensitivity of this protocol. We have also established that PKC-6 is localized in the soluble fraction of rabbit follicles and corpora lutea utilizing homogenization buffers described in Materials and methods (Section 2) [20], therefore only soluble extracts are evaluated for PKC-6 content.

To evaluate when PKC-6 expression was increased in corpora lutea, rabbits were injected with an ovulatory concentration of the LH analog hCG on day 0, some also received an estrogen implant, and luteal PKC-S

protein content and mRNA levels were evaluated on days 1, 2, 3, and 4. Results show that luteal PKC-6 protein (Fig. 1A upper panel) and steady state mRNA levels (Fig. 1C) are increased at least 2-fold by 1 day and reach peak levels by day 2 after hCG injection. The hCG-dependent increase in PKC-6 protein is also seen in a representative autoradiogram showing PKC- 6 autophosphorylation (Fig. 1A middle panel) and a Western blot (Fig. 1A lower panel). The presence of elevated exogenous estrogen did not affect the rate or extent of induction of PKC-6 protein content in corpora lutea during the first 24 h following the induction of ovulation (Fig. 1B). By 4 days after hCG injection, an effect of estrogen on PKC-6 expression is detectable as a 36% increase in PKC-6 content, consistent with the appearance of estrogen responsiveness on days 4 to 6 [3-51 and our previous data [20] showing that estrogen increased PKC-6 expression 2-fold by day 5 of pseudopregnancy.

Based on our evidence that insertion of an estrogen implant at the time of hCG injection resulted in a 2-fold elevation of PKC-6 protein in corpora lutea obtained on day 10 of pseudopregnancy [20], we sought to determine if estrogen also increased steady state levels of PKC-S mRNA. Results demonstrated that indeed estrogen elicited a concomitant and equivalent 2-fold increase in PKC-S mRNA levels (Fig. 2).

We next sought to determine whether estrogen withdrawal on day 9 of pseudopregnancy altered expression of PKC-S protein content and, if results were positive, if this response was reversible. Utilizing the rabbit luteal model developed by Holt et al. [10,13], estrogen was implanted in rabbits at the time of the preovulatory hCG injection. The implants were then either sham removed on day 9 or were removed on day 9 and replaced with blank or estrogen-filled implants on day 11 of pseudopregnancy. PKC-S content was then evaluated in corpora lutea on days 12 to 14 of pseudopregnancy (Fig. 3). Removal of the estrogen implant resulted in a decline in PKC-S with a half- time of approximately 1.75 days (Fig. 3, upper panel). Replacement of the estrogen implant 48 h after removal (i.e., on day 11) resulted in recovery of PKC-6 within 48 h to levels seen in rabbits whose luteal function was maintained by uninterrupted estrogen. Representative serum progesterone levels are reported in Table 1 and are equivalent to those previously reported [10,13].

Finally, we were interested to determine if PKC-S content in corpora lutea varied during pregnancy. We have previously reported that luteal PKC-6 content is equivalent on days 5 and 10 and declines 50% by day 15 of pseudopregnancy. We have now evaluated luteal PKC-F content on days 10, 18, 24 and 28 of pregnancy relative to levels on day 10 of pseudopregnancy. Results in Fig. 4 demonstrate that luteal PKC-6 expression during pregnancy is essentially unchanged but is elevated approximately 60% (P < 0.05) on all days of pregnancy tested compared to that measured in corpora lutea obtained on day 10 of pseudopregnancy.

4. Discussion

We have shown that PKC-F expression is induced in rabbit corpora lutea some 5-fold during the initial 48 h following the induction of ovulation by hCG. Both steady state protein content and mRNA levels appear to be induced concomitantly. Neither the rate nor the extent of hCG-stimulated induction of PKC-6 content by 24 h appears to be affected by the insertion of an estrogen implant at the time of the hCG injection. The inability of exogenous estrogen to modulate expression of PKC-6 in the first 24 h of luteal life is expected since corpora lutea do not exhibit detectable levels of estrogen receptors until days 4-6 of pseudopregnancy [4,5] and develop autonomously in the absence of the ovary or of exogenous estrogen [2]. Indeed, in the rabbit [28] as in most other species, once luteinization has been initiated by an ovulatory concentration of gonadotropin, corpora lutea develop and produce progesterone in the absence of pituitary or ovarian hormone support for the first few days [29]. However, once the rabbit corpus luteum exhibits estrogen receptors and becomes dependent upon estrogen, PKC-6 expression is regulated by estrogen. Both steady state levels of PKC-S protein and mRNA are regulated by estrogen in the corpus luteum of pseudopregnancy. We have demonstrated that exogenous estrogen will increase PKC-6 content by 2-fold on days 5, 10 and 15 of pseudopregnancy [20]. Following the initiation of premature luteal regression upon removal of estrogen implants, exogenous estrogen will also rescue PKC-6 content along with other luteal functions, including progesterone production [15]. The rate of the disappearance of PKC-6 following removal of the es-

Fig. 1. Panel A. Upper panel. PKC-6 content in preovulatory follicles and corpora lutea obtained 1 , 2. 3 and 4 days after the injection of an ovulatory

concentration of hCG to rabbits. PKC-S content was evaluated in IO ug aliquots of soluble extracts of follicles (FOL) or corpora lutea on the

indicated days of pseudopregnancy as “P incorporation into the 76 kDa PKC-S in an in vitro phosphorylation reaction. PKC-S was identified

at 76 kDa by its phosphorylation in the presence of phospholipid and diolein and the absence, but not the presence, of Cal+ [22.27]. “P content

of PKC-S was quantitated on autoradiograms as photostimulable luminescence units (PSL) minus background (BG). Follicles, 1 and 2 day-old

corpora lutea from two rabbits were pooled: otherwise, corpora lutea from a single rabbit were pooled for each experimental data point. Results

are means + S.E.M. of data points indicated at the base of each bar. *Denotes stimulation (P < 0.05) above FOL value. Panel A. Middle panel. Representative autoradiogram showing “P-incorporation into PKC-S in follicles and in corpora lutea on indicated days of pseudopregnancy. Panel

E.T. Maizels et al. I Molecular and Cellular Endocrinology 122 (1996) 213-221 217

--o 3

FOL

*

0 3

1

*

f

3 -t 2

I

Fol Fol 1 1 2 2 4 4

~~-Pcb

WedanbbtofPKc-s

Fd Fd 1 1 4 A

0 I

- CHO-B

DAY OF PSEUDOPREGNANCY

E2 IMPLANT -+ -+

DAYOFPSP 11 44 -

Fig. I.

FOL 1 3

T

t

DAYOF PSEUDOPREGNANCY

A. Lower panel. Western blot of PKC-6 expression in 120 ug aliquots of soluble extracts of follicles and corpora lutea obtained on indicated days of pseudopregnancy. See Materials and methods (Section 2) for experimental details. Panel B. PKC-6 content in corpora lutea obtained I and 4 days after the injection of an ovulatory concentration of hCG and the insertion of an estrogen (Es) implant, as indicated. Results are mean rf: S.E.M. for three data points or means + range of two data points, as indicated. Panel C. PKC-6 mRNA in preovulatory follicles and corpora lutea obtained 1 and 3 days after the injection of an ovulatory concentration of hCG to rabbits. Results show Northern blots for PKC-S and control CHO-B and are expressed as the ratio of PKC-6 to CHO-B (lower bar graph). Follicles from six rabbits and corpora lutea from four rabbits were pooled for each data point. Results are means i range of two data points for corpora lutea on days 1 and 3 of pseudopregnancy.

trogen implant was rather slow, with 40% remaining after 2 days of estrogen withdrawal and low but detectable levels remaining 5 days after estrogen withdrawal. The rescue of PKC-6 protein expression in response to estrogen replacement exhibited a time course which appeared to be generally equivalent to the induction of PKC-6 during luteinization in response to hCG. when PKC-S was detectable within 24 h and maximal levels were reached by 48 h.

That PKC-S expression can be regulated at the mRNA and protein levels both in response to hCG and to estrogen is very interesting and suggests that positive response elements exist on the PKC-6 promoter both for regulatory factors downstream of the estrogen receptor as well as downstream of hCG-stimulated signaling pathways. Cloning of the promoter for PKC-F has not been reported and there are only a few reports addressing the regulation of PKC-6 expression [30,31]. We do not know if PKC-6 expression in the rabbit corpus luteum retains its sensitivity to regulation by hCG once the corpus luteum develops its responsiveness to estrogen. This possibility, however is difficult to test since the injection of an ovulatory concentration of hCG into pseudopregnant or pregnant rabbits leads to luteal regression, characterized by a decline in serum progesterone and luteal weight [I 11, a response due in part to the elimination of follicular estrogen since these responses can be reversed upon insertion of an estrogen implant [32]. However, it would be interesting to determine if low concentrations of hCG affect expression of PKC-6 in the rabbit corpus luteum.

1 .oc I-

0.80

0.60

0.40

0.20 -cl 5

T

CON E2

1:

+

6

:

Effect of estrogen on steady state PKC-d mRNA levels m

corpora lutea obtained from IO-day pseudopregnant rabbits. Rabbits

were injected with an ovulatory concentration of hCG and untreated

(CON) or given an estrogen implant (EZ) on day 0. Corpora lutea were pooled from a single animal for each data point. Results are

means i S.E.M. from 5 and 6 data points, as indicated, of the ratio

of PKC-?i to CHO-B. *Denotes stimulation (P < 0.05) above control

level.

lE4 -c

i+i 8000~- t-! Pi? 5 +.- 6000-- vz QE

2 z 4000-- no

a

= 2000--

l -.CONTlNUOUS E2

T o--oWITHDRAWN

A.- - - A E2 REPLACEMENT

0-I 8 9 10 11 12 13 14

DAY OF PSEUDOPREGNANCY

10 10 IO 14 14 14 14 14 14 14 14 DayofPSP

- PKC-6

0+10 o-)9 O-09 0+9 1 E; implant 9 +14 11+14

Fig. .J. Upper panel. Effect of estrogen withdrawal on PKC-6 expres-

sion in rabbit corpora lutea. An estrogen implant was inserted on the

day of the ovulatory hCG injection (day 0). On day 9 of pseudopreg-

nancy. implants were left in place (continuous E,, 0) or were with-

drawn for 48 h. On day 1 I of pseudopregnancy in estrogen

withdrawn rabbits, either blank (,-b) or estrogen-filled (A) implants

were replaced. Corpora lutea were obtained on days 9. I I. 12. 13. and

14 of pseudopregnancy, as indicated. PKC-F content in soluble luteal

extracts was evaluated as described in the legend to Fig. IA. Results

are means + S.E.M. of three rabbits for all points except for corpora

lutea obtained on day 14 from rabbits receiving a blank implant on

day I 1. where results are means + range of two rabbits. *Denotes

difference (P < 0.05) from value in corpora lutea obtained on day 1 I ol’ pseudopregnancy from rabbits withdrawn from estrogen for 48 h.

Values denoted by * are not different (P > 0.05) from each other.

Value in corpora lutea obtained on day 12 of pseudopregnancy from

rabbits withdrawn from estrogen for 48 h and replaced with estrogen

for 24 h (A) is not different (P > 0.05) from value in corpora lutea

obtained on day 1 I of pseudopregnancy in rabbits receiving continu-

ous estrogen. Lower panel. Representative autoradiogram showing

“P-incorporation into PKC-F. The presence of an estrogen implant

on indicated days of pseudopregnancy (PSP) is denoted.

Once PKC-6 is induced with luteinization in rabbit corpora lutea, PKC-6 protein content appears to be maintained at relatively steady levels throughout pseu-

dopregnancy [20] and pregnancy until luteal demise, although levels in corpora lutea of pregnancy are approximately 60% higher than levels on day 10 of pseudopregnancy. This pattern of regulation of PKC-S expression is in striking contrast to its regulation in the rat corpus luteum. PKC-K protein is expressed at rela-

E.T. Maizels et al. / Molecular and Cellular Endocrinology 122 (1996) 213-221 219

tively low levels in corpora lutea obtained throughout the luteal life span of hCG-induced pseudopregnancy in immature rats and is barely detectable in corpora lutea of pregnant rats until the second half of pregnancy, when levels rise at least lo-fold to peak values 4 to 5 days prior to parturition [27]. Thus, in the rat corpus luteum PKCd expression is not stimulated by the preovulatory surge of gonadotropin that promotes ovulation. In fact, if anything, PKC-6 expression in rat corpora lutea formed following a fertile mating is re- duced compared to levels detected in preovulatory folli- cle-enriched ovaries [27]. However, like the rabbit corpus luteum, estrogen positively regulates PKC-6 expression in rat corpora lutea [27].

The function of PKC-6 in the rabbit corpus luteum is not known. It is unlikely that PKC-S is associated with progesterone production since the estrogen-stimulated increase in PKC-6 expression in corpora lutea of rabbits is not associated with a concomitant increase in serum progesterone levels [8,10,12]. Consistent with its ubiquitous distribution [33-351 and the resulting likeli- hood of its involvement in fundamental cellular functions, recent studies indicate that PKC-6 is important in the negative regulation of cell proliferation [36-391. Since luteal cells are not mitogenic, it is likely that PKC-6 subserves additional functions in corpora lutea unrelated to cell growth.

We also do not know which hormones or growth factors acutely modulate the activity of PKC-F in the rabbit corpus luteum. Likely candidates for the activation of PKC-6 in the rabbit corpus luteum include epidermal growth factor and platelet derived growth factor, both of which have been shown to promote the selective activation of PKC-8 in other cells [40-421, as well as insulin-like growth factor-l, which can also activate PKCs [43] and has been shown to modulate

Table 1 Effect of removal of estrogen-filled implant on serum progesterone levels on days 11 to 14 of pseudopregnancy

Treatment Serum progesterone levels” (ngiml, n)

Continuous estrogen day O-14 Estrogen implant replaced on

day 12

14.94 & 3.28b* (5) 8.77 k 1.36=* (6)

Estrogen implant not replaced on day 12

1.90 + 0.53d* (6)

*Denotes values which are different (PcO.05). “Results are means + S.E.M. of (n) rabbits of serum progesterone levels measured on the indicated days of pseudopregnancy. “Progesterone levels were measured in serum obtained on days 11 (2 rabbits) and 14 (3 rabbits). ‘Progesterone levels were measured in serum obtained on days 13 (3 rabbits) and 14 (3 rabbits). dProgesterone levels were measured in serum obtained on days 11 (3 rabbits) and 14 (3 rabbits).

7500

0

-E, +& 10 18 24 28 ------ Day 10 of PSP

IL 2

Oay of Pregnancy

I 2

T

1 2

DlO PSP

10 18 24 28

DAY OF PREGNANCY

Fig. 4. PKCd content in soluble extracts of corpora lutea obtained on indicated days of pseudopregnancy or pregnancy in the rabbit. Upper panel. Representative autoradiogram showing 32P incorporated into PKC-6 on indicated days of pregnancy and pseudopregnancy. PKC-6 in corpora lutea of lo-day pseudopregnant rabbits given an estrogen implant on day 0 is shown for reference. Lower panel. PKC-8 content was evaluated as described in the legend to Fig. IA. Results are means If: range of two rabbits or mean k S.E.M. of three rabbits, as indicated. Luteal PKC-S content during pregnancy is not different (P > 0.05) as determined by analysis of variance; luteal PKC-6 content on day 10 of pseudopregnancy is different (P < 0.05) from all days of pregnancy tested, as determined by analysis of variance and Duncan’s multiple range test.

luteal function in the rabbit [44]. In addition to poten- tial regulation during luteal life, PKC-F may also be selectively activated by luteolytic signals/responses like prostaglandin F2cl and tumor necrosis factor-a, both of which can activate PKCs [45,46] and are involved in luteal demise in the rabbit [47,48].

In summary, we have shown that PKC-8 protein and steady state mRNA levels are induced 5-fold in corpora lutea by the preovulatory gonadotropin surge that induces ovulation and luteinization and that PKC-F protein and mRNA levels are then maintained in corpora lutea throughout pseudopregnancy by estrogen. Although its induction with corpus luteum formation and maintenance throughout the luteal life span suggests that PKC-6 is important to the normal function of the rabbit corpus luteum, additional studies are needed to understand the role of this specific PKC isotype in luteal function.

220 E.T. Maizrls et 01. / Molecular und Cellulur Ehdocrinology 122 (1996) 213-221

Acknowledgements [I71

We would like to thank Dr. Peter J. Parker for providing us with the cDNA fragment of PKC-6 and Drs. Shigeo Ohno and Keiko Mizuno for providing us with antisera to PKC-6.

[‘81

[I91

References

PI

[21

[31

[41

[51

161

t71

PI

[91

UOI

PII

u21

[I31

1141

1151

Holt. J.A. (1989) Regulation of progesterone production in the

rabbit corpus luteum. Biol. Reprod. 40, 201~ 208.

Miller, J.B. and Keyes. P.L. (1975) Progesterone synthesis in

developing rabbit corpora lutea in the absence of follicular

estrogens. Endocrinology 97. 83 - 90.

Miller, J.B. and Keyes, P. L. (I 978) Transition of the rabbit corpus

luteum to estrogen dependence during early luteal development.

Endocrinology 102. 31-38.

Mills. T.M. and Osteen, K.G. (1977) 17P-Estradiol receptor and

progesterone and 20a-hydroxy-4-pregnen-3-one content of the

developing corpus luteum of the rabbit. Endocrinology 101.

174& 1750.

Miller. J.B. and Toft, D.O. (I 983) Characterization and physiolog-

ical variation of estrogen receptors in rabbit corpora luteum

throughout pregnancy and pseudopregnancy: the effect of hysterec-

tomy and sustained estradiol treatment. Biol. Reprod. 29.620-629.

Yuh, K.-C.M. and Keyes, P.L. (1979) Properties of nuclear and

cytoplasmic estrogen receptor in the rabbit corpus luteum: evidence

for translocation. Endocrinology 105. 690.-696.

Drake, R.G. and Cook, 8. (1979) The estrogen receptor of rabbit

corpus luteum: binding, dissociation and stability characteristics.

Endocrinology 105. 561-569.

Bill I1,C.H. andKeyes, P.L. (1983) I’@-Estradiolmaintainsnormal

function of corpora lutea throughout pseudopregnancy in hypo-

physectomized rabbits. Biol. Reprod. 28, 608-617.

Holt, J.A.. Dinerstein, R.J. and Lorincz. M.A. (1982) Site of

estrogen action in rabbit ovarian tissue. Am. J. Physiol. 243.

E188~E195.

Holt, J.A., Keyes, P.L., Brown. J.B. and Miller, J.B. (1975)

Premature regression of corpora lutea in pseudopregnant rabbits

following removal of polydimethylsiloxane capsules containing

l7@-estradiol. Endocrinology 97. 76-82.

Day. S.L. and Birnbaumer. L. (1980) The effect of estradiol on

hormonally stimulable adenylyl cyclase activity and on proges-

terone production in normal and regressing corpora lutea from

control and human chorionic gonadotropin-treated pseudopreg-

nant rabbits. Endocrinology 106. 375-381.

Dharmarajan. A.M.. Zanagnolo. V.L., Dasko. L.M., Zirkin, B.R..

Ewing, L.L. and Wallach, E.E. (I 991) Estradiol regulation of the

rabbit corpus luteum: in vivo and in vitro studies. Endocrinology

128, 2678-2684.

Bender, E.M.. Miller, J.B., Possley. R.M. and Keyes, P.L. (1978)

Steroidogenic effect of l7P-estradiol in the rabbit: stimulation of

progesterone synthesis in prematurely regressing corpora lutea.

Endocrinology 103, 1937-1943.

Holt, J.A., Lorincz, M.A. and Lyttle, C.R. (1981) Estrogen receptor

in rabbit ovaries and effects of antiestrogen on progesterone production. Endocrinology 108. 3308-2315.

Keyes, P.L., Kostyo, J.L.. Hales, D.B., Chou, S.Y., Constantino,

C.X. and Payne, A.H. (1990) The biosynthesis of cholesterol

side-chain cleavage cytochrome P-450 in the rabbit corpus luteum

depends upon estrogen. Endocrinology 127, II 86- 1193.

[I61 Holt. J.A., Lorinca. M.A. and King, W.J. (1983) Monoclonal

antibody recognition of estrogen receptor in rabbit corpus luteum. Am. J. Physiol. 7, E494-E498.

PO1

PII

PI

1231

v41

~251

WI

~271

VI

1291

WI

[311

~321

[33]

[341

[351

Holt. J.A. and Schreiber, J.R. (1985) Estrogen regulation of

steroidogenesis in rabbit luteal cells. Endocrinology 117. 2462- 2470.

Wittmaack, F.M., Holt, J.A. and Schreiber, J.R. (1986)Cholesterol

metabolism in estrogen-sensitive progestin synthesis by rabbit

corpus luteum. Am. J. Physiol. 251, E457-E463.

Holt. J.A., Wittmaack. F.J., Schreiber, J.R., Ghosh, D.K. and

Menon, K.M.J. (1988) Estrogen increases precursor for preg- nenolone synthesis with temperature-sensitive occupancy of P-

450,,, in mitochondria of rabbit corpus luteum. Endocrinology 122,

1948-1957.

Maizels, E.T., Miller, J.B., Cutler Jr, R.E., Jackiw. V., Carney,

E.M., Mizuno, K., Ohno, S. and Hunzicker-Dunn, M. (1992)

Estrogen modulates Ca’ +-independent lipid stimulated kinase in

the rabbit corpus luteum of pseudopregnancy. J. Biol. Chem. 267.

17061 -17068.

Dekker. L.V. and Parker. P.J. (1994) PKC: aquestionofspecificity.

TIBS 49. 73-77.

Hug, H.. and Sarre, T.G. ( 1993) PKC isotypes: divergence in signal

transduction. Biochem. J. 291, 329-343.

McFadden, P.N.. Nandpe, A. and Koshland Jr. D.E. (1989)

Calcium- and lipid-independent protein kinase C autophosphory-

lation. Activation by low pH. J. Biol. Chem. 264, 12765-12771.

Lowry. O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.

(I 95 I ) Protein measurement with the Folin phenol reagent. J. Biol.

Chem. 193, 265-275.

Maizels. E.T., Miller, J.B.. Cutler Jr, R.E., Jackiw, V., Carney.

E.M.. Kern, L. and Hunzicker-Dunn, M. (1990) Calcium-indepen-

dent phospholipid/dioiein-dependent phosphorylation ofa soluble

ovarian M, 80 000 substrate protein: biochemical characteristics.

Biochim. Biophys. Acta 1054. 285-296.

Cutler Jr. R.E., Maizels. E.T., Brooks, E.J.. Mizuno, K., Ohno.

S.. and Hunzicker-Dunn, M. (1993) Regulation of 6 protein kinase

C during rat ovarian differentiation. Biochim. Biophys. Acta 1179.

260.. 270.

Cutler Jr, R.E., Maizels, E.T. and Hunzicker-Dunn. M. (1994) 6

Protein kinase-C in the rat ovary: estrogen regulation and local-

ization. Endocrinology 135, 1669.- 1678.

Yuh, K.-C.M.. Bill II, C.H. and Keyes, P.L. (1984) Transient

development and function of rabbit corpora lutea after hypophy-

sectomy. Am. J. Physiol. 247, E808-E814.

Rothchild, I. (1981) The regulation of the mammalian corpus

luteum. Rec. Prog. Horm. Res. 37. 183-298.

Kim, H.-M.. Hirota, S., Onoue. H.. Hirata, T., Suzuki. K., Ohno,

S., Kuroki. T., Kitamura, Y. and Nomura, S. (1992) Localization

and developmental expression of a novel protein kinase C 6 gene.

Dev. Brain Res. 70, 239-244.

Jun, C.-D., Choi. B.-M.. Lee. S.-Y.. Kang. S.-S., Kim, H.-M. and

Chung, H.-T. (1994) Nitric oxide inhibits the expression of protein

kinase C 6 gene in the murine peritoneal macrophages. Biochem.

Biophys. Res. Commun 204, 105- 1 Il. Yuh. K.-C.M. and Keyes, P.L. (1981) Effects of human chorionic

gonadotropin in the rabbit corpus luteum: loss ofestrogen receptor

and decreased steroidogenic response to estradiol. Endocrinology

108, 1321. 1327.

Mizuno. K., Kubo, K.. Saido. T.C., Akita, Y., Osada, S.. Kuroki.

T.. Ohno. S. and Suzuki. K. (I991 ) Structure and properties of a

ubiquitously expressed protein kinase C, nPKC 6. Eur. J. Biochem. 202, 931-941.

Leibersperger, H., Gschwendt. M.. Gernold, M. and Marks, F.

( 1991) Immunological demonstration of a calcium-unresponsive

protein kinase C of the S-type in different species and murine tissues.

Preponderance in epidermis. J. Biol. Chem. 266. 14778-14784.

Wetsel. W.C., Khan, W.A., Merchenthaler. I., Rivera, H., Halpern.

A.E.. Phung. H.M., Negro-Vilar, A. and Hannun. Y.A. (1992)

Tissue and cellular distribution of the extended family of protein kinase C isoenzymes. J. Cell. Biol. 117, 121-133.

E.T. Maizefs et al. 1 Molecular and Cellular Endocrinology 122 (1996) 213-221 221

[36] Mischak, H., Goodnight, J., Kokch, W., Martiny-Baron, G., Schaechtle, C., Kazanietz, M.G., Blumburg, P.M., Pierce, J.H. and Mushinski, J.F. (1993) Over-expression of PKC-6 and - E in NIH 3T3 cells induces opposite effects on growth, morphology, anchor- age dependence, and tumorigenicity. J. Biol. Chem. 268, 6090- 6096.

[37] Watanabe, T., Ono, Y., Taniyama, Y., Hazama, K., Igarashi, K., Ogita, K., Kikkawa, U. andNishizuka, Y. (1992)Celldivisionarrest induced by phorbol ester in CHO cells overexpressing PKC-6 subspecies. Proc. Natl. Acad. Sci. USA 89, 10159-10163.

[38] Mishima, K., Ohno, S., Shitara, N., Yamaoka, K. and Suzuki, K. (1994) Opposite effects of the over-expression of PKC-y and 6 on the growth properties of human glioma cell line U251 MCI. B&hem. Biophys. Res. Commun. 201, 363-372.

[39] Hirai, S., Izumi, Y., Higa, K., Kaibuchi, K., Mizuno, K., Osada, S., Suzuki, K. and Ohno, S. (1994) Ras-dependent signal transduction is indispensable but not sufficient for the activation ofAP1 /Jun by PKC-6. EMBO J. 13, 2331-2340.

[40] Ohno, S., Mizuno, K., Adachi, Y., Hata, A., Akita, Y., Akimoto, K., Osada, S.-I., Hirai, S.-i., and Suzuki, K. (1994) Activation of novel protein kinases C6 and Cc upon mitogenic stimulation of quiescent rat 3Yl fibroblasts. J. Biol. Chem. 269, 17495517501.

[41] Xu, J., Rockow, S.. Kim, S., Xiong, W. and Li, W. (1994) lnterferons block protein kinase C-dependent but not -independent activation of raf-1 and mitogen-activated protein kinases and mitogenesis in NIH 3T3 cells. Mol. Cell. Biol. 14, 8018-8027.

[42] Li, W., Yu, J.-C., Michieli, P., Beeler, J.F.. Ellmore, N., Heidaran, M.A. and Pierce, J.H. (1994) Stimulation of the platelet derived growth factor B receptor signaling pathway activates protein kinase C-6. Mol. Cell. Biol. 14, 6727-6735.

[43] Heidenreich, K.A., Zeppelin, T. and Robinson, L.F. (1993) Insulin and insulin-like growth factor I induce c-fos expression in postmi- totic neurons by a protein kinase C-dependent pathway. J. Biol. Chem. 268, 14663-14670.

[44] Constantino, C.X., Keyes, P.L. and Kostyo, J.C. (1991) Insulin-like growth factor I stimulates steroidogenesis in rabbit luteal cells. Endocrinology 128, 1702- 1708.

[45] Gorospe, M., Kumar, S. and Baglioni, C. (1993) Tumor necrosis factor increases stability of interleukin-1 mRNA by activating protein kinase C. J. Biol. Chem. 268, 6214-6220.

[46] Niswender, G.D., Juengel, J.L., McGuire, W.J., Belfiore, C. J. and Wiltbank, M.C. (1994) Luteal function: the estrous cycle and early pregnancy. Biol. Reprod. 50, 2399247.

[47] Miller, J.B. and Pawlak, C.M. (1993) Characterization and physiological variation in prostaglandin, prostacyclin and thromboxane synthesis by corpora lutea, non-luteal and uterine tissues during pseudopregnancy in the rabbit. Life Sci. 54, 341-353.

[48] Bagavandoss, P., Wiggins, R.C., Kunkel, S.L., Remick, D.G. and Keyes, P.L. (1990) Tumor necrosis factor production and accumulation of inflammatory cells in the corpus luteum of pseudopregnancy and pregnancy in rabbits. Biol. Reprod. 42, 3677376.