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a Institute of Medical Biochemistry,
b Department of Tumor Biology,
c Norwegian Radium Hospital, and Neurochemical Laboratory, University of Oslo, 0317 Oslo, Norway
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, ß, and 
) were detected. The expression levels of mRNAs for PKC and PKCß were also up-regulated (2.5- to 3-fold) by TPA (10-7 M), but these effects were much slower (peaking after 12 h) than those on phosphotransferase activity. In the presence of TPA (10-7 M), expression of androgen receptor (AR) mRNA showed a transient time-dependent down-regulation (~70%), in which the nadir was reached after 6 h and baseline expression was again obtained after 12 h. The regulatory effect of PKC activation on AR mRNA was confirmed by the absence of response to a biologically inactive phorbol ester. A concentration-dependent decrease (half-maximal effect at ~10-8 M TPA) of AR mRNA was also observed. These data suggest that Sertoli cell responses to testosterone may be inhibited by a transiently active PKC with a wide intracellular distribution.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Sertoli cells, receptor-mediated hydrolysis of phosphoinositides by activation of phospholipases may constitute another important transducing mechanism for extracellular stimuli [6, 7]. Various studies have demonstrated that regulatory responses governed by the calcium/phospholipid-dependent protein kinase (protein kinase C; PKC), the major receptor for tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) [8], may abrogate cAMP-dependent effector mechanisms in Sertoli cells. Treatment of Sertoli cells with TPA has been shown to antagonize FSH-stimulated cAMP production, probably through phosphorylation of the FSH receptor [9], as well as to up-regulate the regulatory subunits type I and, in particular, type IIß of the cAMP-dependent protein kinase, presumably leading to lower catalytic activity of this enzyme [10]. Consequently, TPA is considered to influence cAMP-dependent physiological responses such as aromatization of testosterone, which is inhibited [9].
The regulatory effects of FSH and cAMP analogues on AR expression [11–13] and transcriptional activity [14] may also contribute to the complexity of intracellular signal "cross-talking" in Sertoli cells. The possibility that Sertoli cell responses to testosterone also are under the control of signal transduction events activating PKC, whether converging with cAMP-dependent effector mechanisms or not, is particularly appealing.
The present study was performed to characterize one aspect of the calcium/phospholipid-dependent signaling pathway in cultured rat Sertoli cells. We examined activation of PKC and regulatory effects on mRNA expression of the AR to evaluate whether this kinase may modulate Sertoli cell responses governed by testosterone.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sertoli cells were isolated from 19-day-old Sprague-Dawley rats (Møllegaard's Breeding Centre Ltd., Copenhagen, Denmark) as previously described [15, 16]. The cells were plated in Minimum Essential Medium Eagle (MEM; Gibco BRL, Middlesex, UK) containing 10% fetal calf serum (Gibco BRL) and L-glutamine (2.0 mM; Gibco BRL) in 5% CO2 at 34°C. After 72 h, the culture medium was replaced by serum-free MEM, and incubations were continued for another 48 h before the start of experimental incubations. At the start of each experiment, the culture medium was exchanged for fresh MEM containing TPA, the cAMP analogue 8-(4-chlorophenylthio-)cAMP (8-CPTcAMP), or the biologically inactive phorbol ester 4-phorbol, all obtained from Sigma (St. Louis, MO). The care of animals and the experimental protocol were reviewed and approved by the animal care committee of our institution.
Isolation of Subcellular Fractions and Phosphotransferase Assay
After the experimental incubations, the Sertoli cells were harvested and homogenized in ice-cold buffer containing 0.25 M sucrose, 10 mM Tris-HCl (pH 7.4), 2 mM EGTA, 2 mM EDTA, 0.1% ß-mercaptoethanol, 100 µM PMSF, and 10 µg/ml each of leupeptin, antipain, chymostatin, and peptatin. Soluble and particulate fractions were separated by centrifugation at 30 000 x g, and the pellets (particulate fractions) were solubilized by resuspension in homogenization buffer containing 1% Triton X-100. Aliquots from the subcellular fractions (containing 1–3 µg protein) were incubated in a volume of 100 µl containing (final concentrations) 10 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 50 µM [-32P]ATP (Amersham Laboratories, Buckinghamshire, UK), 0.01% ß-mercaptoethanol, 2 µM Walsh inhibitor, and 0.25 M HEPES (pH 7.4), in the absence or presence of 1.5 mM CaCl2 and phosphatidylserine (1 mg/ml). Histone IIIS (10 µg/reaction) was added as substrate, and the tubes were preincubated at 30°C for 30 sec before reactions were initiated by addition of ATP. After incubation at 30°C for 5 min, the reactions were terminated by boiling with Laemmli sample buffer. Phosphoproteins were separated by SDS-PAGE, and dried gels were visualized by autoradiography. The data referred to were reproduced in three independent experiments.
Northern Blot Analyses
Total RNA was extracted and analyzed by standard Northern blot technique. Samples of 20 µg RNA were resolved by gel electrophoresis before transfer onto BioTrans nylon membranes (ICN, Irvine, CA). Equal loading in each lane was verified by ethidium bromide staining. The cDNAs for PKC isozymes were bovine fragments [17] of PKC, PKCß, and PKC. The rat AR cDNA [18] contained the 3'-part of the open reading frame (representing exons 4–8). The probe for the ribosomal factor L27 was purchased from the American Type Culture Collection (Rockville, MD). The cDNA probes were labeled with [-32P]dCTP (Amersham Laboratories) by the random priming technique, and standard hybridization conditions (50% formamide, 42°C) were used. Final autoradiographs were subjected to densitometric readings in a Molecular Dynamics 300A laser densitometer (Sunnyvale, CA), and the values obtained were normalized to the corresponding intensities of L27 mRNA signals. The data shown were reproduced in at least three independent experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sertoli cells were incubated with TPA (10-7 M) for increasing time periods (5 min to 24 h), and phosphotransferase activity in the absence and presence of calcium and phosphatidylserine was analyzed in soluble and particulate cell fractions. Figure 1, displaying the enzymatic profile from the particulate cell fraction, shows low constitutive PKC activity. Treatment with TPA was associated with a transient induction of calcium/phosphatidylserine-dependent activity, which was much higher than in the absence of these cofactors. Peak enzyme activity was detected between 5 min and 1 h of TPA treatment, followed by a gradual decline to baseline after 24 h. An essentially similar profile of phosphotransferase activity was obtained in the soluble cell fraction (not shown).
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Consequently, constitutive expression of mRNAs for the calcium/phospholipid-dependent isomeric forms of PKC (, ß, and ) in the Sertoli cells was analyzed. As seen from Figure 2, mRNAs for the universally expressed PKC (8.5-kilobase [kb] and 3.9-kb bands) as well as for PKCß (9.5-kb, 3.3-kb, and 2.8-kb bands) were detected. Surprisingly, three specific transcripts for PKC mRNA, an isotype considered to be restricted to neural crest-derived cell types [19, 20], were also found, including a putative novel mRNA species (smaller than the 3.5-kb and 3.1-kb bands).
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Moreover, Sertoli cells were incubated with TPA (10-7 M) for increasing time periods (0–24 h) to examine possible regulatory effects on PKC expression (Fig. 3). Regulation of the expression level turned out to be much slower than regulation of phosphotransferase activity. The expression of PKC mRNA in TPA-treated cells was not different from that in control cells before 6–12 h of incubation, when an up-regulation (~3-fold) was seen. A TPA-dependent increase in PKCß mRNA was also observed, although the amplitude of this response appeared to be somewhat lower and even more delayed (~2.5-fold after 12 h of incubation). The mRNA expression level of PKC was low and essentially unresponsive to TPA (not shown). The mRNA encoding the ribosomal protein L27 is depicted to verify equal loading and specificity of the TPA responses (Fig. 3).
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Effects of PKC Activation on AR mRNA Expression
Next, possible functional effects of PKC activation in Sertoli cells were analyzed. First, changes in AR mRNA expression were measured in Sertoli cells treated with TPA (10-7 M) for 1–12 h (Fig. 4). The level of AR mRNA revealed a transient decrease (~70%) at 6 h, followed by a gradual return to the baseline level after 12 h. Similarly, a concentration-dependent decline in AR mRNA expression was observed at 6 h (Fig. 5). Whereas baseline expression was close to unresponsive at low concentrations of TPA (10-10–10-9 M), higher concentrations (10-8–10-6 M) were associated with a reduction in the level of AR mRNA, with a half-maximal effect at 10-8 M TPA and a maximal reduction (~70%) at the highest concentrations. Analysis of mRNA expression of PKC and PKCß was also included (Fig. 5). In accordance with the data displayed in Figure 4, PKC mRNA was significantly induced (3- to 4-fold) in the presence of the higher concentrations of TPA (10-8–10-6 M), whereas PKCß mRNA was not (i.e., induced by < 2-fold).
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The Sertoli cell AR has previously been shown to be transiently down-regulated by FSH or cAMP analogues [12, 13]. To analyze the specificity of the PKC-dependent regulatory pathway, the effect of TPA (10-7 M for 6 h) on AR mRNA was compared with responses to treatment for 6 h with the cAMP analogue 8-CPTcAMP (3 x 10-5 M) as well as the biologically inactive phorbol ester 4-phorbol (10-7 M). As shown by Figure 6 and consistent with earlier reports [12, 13], AR mRNA was found to be significantly down-regulated (~90%) by 8-CPTcAMP. A TPA-dependent decrease (~65%) in AR mRNA expression was again observed, whereas 4-phorbol was essentially inactive.
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In all analyses, L27 mRNA is shown as an internal control (Figs. 4–6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, ß, and ). Activation of PKC was associated with homologous induction of mRNA expression of PKC as well as a transient inhibitory effect on AR mRNA expression.
The kinetics of the TPA-dependent regulation of PKC expression appeared to be much slower than that of phosphotransferase activity and also somewhat delayed when compared to the regulatory effect on AR mRNA. These observations indicate that homologous regulation of PKC expression levels may represent a homeostatic control mechanism or principally control long-term regulatory mechanisms but is presumably not crucial in Sertoli cell processes that are governed by testosterone. The concept of specific down-regulation of AR mRNA by kinase activation was also supported by the apparent absence of regulatory effect of the biologically inactive phorbol ester 4-phorbol.
The classical PKC isotypes (, ßI, ßII, and ) are biochemically characterized by their in vitro activation by calcium, phosphatidylserine, and diacylglycerol (reviewed in [21, 22]) or its analogue TPA [8]. The PKC isozymes reveal uneven tissue distribution. Some are widespread, such as PKC and PKCßII, whereas PKC has been considered to be restricted to neural crest-derived cell types [19, 20]. Our data demonstrated a novel physiological aspect with Sertoli cell PKC since, in addition to mRNAs for the and ß isotypes, PKC mRNA was also expressed. Although it is difficult to extrapolate from the measurement of an mRNA to the function of a protein, the PKC mRNA may represent the only non-neural-related localization of this PKC isoform so far reported. Analysis of testicular function in the recently generated PKC knock-out mouse model [20] would be particularly interesting in this regard.
Moreover, in a variety of cells and tissues studied, the bulk of PKC has shown rapid redistribution from cytoplasm to the particulate cell fraction upon activation, correlating with the course of agonist-induced activation of PKC by mobilization of phospholipase-dependent signaling pathways [21, 22]. As was the case for membrane-associated phosphotransferase activity, TPA-dependent PKC activity in Sertoli cells was also present in the soluble cell extracts. Hence, activation and desensitization of the TPA response seemed to follow the kinetics of classical PKC isotypes in both Sertoli cell fractions. Others have shown that the ratio of basal PKC activity in the soluble versus the particulate cell fraction of Sertoli cells increases at the onset of pubertal maturation, arguing that this represents a pattern of intracellular PKC distribution that is typical of nonproliferating, differentiated cells [23].
The Sertoli cell AR is regulated by several hormonal factors. Transient inhibition of AR mRNA expression is observed after treatment with FSH or cAMP analogues [12, 13], whereas long-term induction of the cAMP-dependent protein kinase results in a significant up-regulation of the level of AR mRNA [11–13]. Our observation that PKC activation was associated with transient down-regulation of AR mRNA implies that cAMP-dependent and PKC-activated effector mechanisms may converge along the intracellular signaling cascades or on the level of target gene regulation. A functional cAMP response element as well as an activating protein-1 (AP-1) binding motif, representing a TPA response element, have been demonstrated in the promoter region of the human AR gene [24], but the corresponding region of the rat gene has not been characterized [24, 25]. The transient down-regulation by FSH of AR mRNA expression in rat Sertoli cells, however, has been explained by a decrease in AR mRNA stability [12]. Whether PKC activation also increases the rate by which AR mRNA is degraded is not known.
It is generally believed that FSH synergizes the effect of testosterone on spermatogenesis, for which both factors are probably required [4, 5]. Interestingly, recent data strongly indicate that if the action of FSH is missing, normal testicular production of testosterone can compensate for this to maintain human fertility [26]. Our data indicate that PKC activation antagonizes testosterone action in pubertal Sertoli cells. The inhibitory effect on AR expression might suggest that PKC activation represents an independent compensatory mechanism that desensitizes Sertoli cells to androgen-dependent processes. Hydrolysis of phosphoinositides in Sertoli cells is elicited by different extracellular stimuli, such as purine nucleotides [6] and vasoactive hormones like angiotensin II or vasopressin [7], as well as the presence of germ cells in coculture with the Sertoli cells [27, 28]. Several studies have demonstrated germ cell modulation of Sertoli cell response to FSH [28–30], and in vivo the production of secretory proteins by Sertoli cells is controlled by the adjacent germ cell population [31, 32]. Recent reports have shown that the expression level of rat Sertoli cell AR is both stage-dependent (of the spermatogenic cycle) and age-dependent [33–35], consistent with the suggestion that haploid germ cells are involved in the local control of AR expression. Thus, the possibility that the PKC signaling pathway in Sertoli cells operates within a paracrine mechanism that functionally governs the spermatogenic process is particularly appealing but must be proven in a physiological context, e.g., seminiferous tubules of adult animals.
To summarize, the present study has characterized a novel aspect of the PKC signaling pathway in cultured Sertoli cells isolated from immature rats. Activation of PKC, displaying the kinetics of classical isotypes and present both in the soluble and particulate cell fractions, was associated with a transient inhibition of AR mRNA. These data suggest that PKC activation in Sertoli cells might represent an independent regulatory mechanism in spermatogenesis, presumably by intracellular "cross-talking" with the signal transduction pathway of testosterone.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Anne Hansen Ree, Department of Tumor Biology, Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway. FAX: 47 2252 2421; ahree{at}radium.uio.no
3 Current address: Winnie Eskild, Department of Biochemistry, University of Oslo, Oslo, Norway.
Accepted: December 29, 1998.
Received: December 29, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) via multiple and distinct mechanisms. Endocrinology 1992; 130:1271–1280.. Science 1997; 278:279–283.This article has been cited by other articles:
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  T. Saether, T. N. Tran, H. Rootwelt, B. O. Christophersen, and T. B. Haugen Expression and Regulation of {Delta}5-Desaturase, {Delta}6-Desaturase, Stearoyl-Coenzyme A (CoA) Desaturase 1, and Stearoyl-CoA Desaturase 2 in Rat Testis Biol Reprod, July 1, 2003; 69(1): 117 - 124. [Abstract] [Full Text] [PDF]
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K. W. Braun, M.-N. Vo, and K. H. Kim Positive Regulation of Retinoic Acid Receptor Alpha by Protein Kinase C and Mitogen-Activated Protein Kinase in Sertoli Cells Biol Reprod, July 1, 2002; 67(1): 29 - 37. [Abstract] [Full Text] [PDF]
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 J. k. Chen and L. L. Heckert Dmrt1 Expression Is Regulated by Follicle-Stimulating Hormone and Phorbol Esters in Postnatal Sertoli Cells Endocrinology, March 1, 2001; 142(3): 1167 - 1178. [Abstract] [Full Text] [PDF]
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