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PROTEIN KINASE C PATHWAY IS INVOLVED IN REGULATING THE SECRETION OF PROSTATIC ACID PHOSPHATASE IN HUMAN PROSTATE CANCER CELLS
Cell Biology International 2001, Vol. 25, No. 11, 1139–1148 doi:10.1006/cbir.2001.0794, available online at http://www.idealibrary.com on
PROTEIN KINASE C PATHWAY IS INVOLVED IN REGULATING THE SECRETION OF PROSTATIC ACID PHOSPHATASE IN HUMAN PROSTATE CANCER CELLS MING-FONG LIN*†, XIU-QING ZHANG, JEANENNE DEAN and FEN-FEN LIN Department of Biochemistry/Molecular Biology, 1Section of Urologic Surgery, and 1Eppley Cancer Institute, University of Nebraska Medical Center, and 1Omaha VA Medical Center, Omaha, NE 68198, U.S.A. Received 17 January 2001; Accepted 7 June 2001 The stimulated secretion of prostatic acid phosphatase (PAcP) has been known to be a hallmark of androgen action on human prostate epithelial cells for the last five decades. The molecular mechanism of androgen action on PAcP secretion, however, has remained mostly unknown. We investigated the molecular mechanism that promotes PAcP secretion in LNCaP human prostate carcinoma cells which express PAcP and are androgen-responsive. Treatment with 12-o-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase C (PKC) activator, resulted in an increased secretion of PAcP in a dose- and time-dependent fashion. 4-Phorbol, a biologically inactive isomer of TPA, had no effect. This TPA stimulation of PAcP secretion was observed 2 h after exposure, while TPA did not have a significant effect on the mRNA level even with a 6 h treatment. A23187 calcium ionophore, known to mobilize cellular calcium which is a co-factor of PKC, also activated PAcP secretion. This TPA stimulation of PAcP secretion was more potent than the conventional stimulating agent 5-dihydrotestosterone (DHT) at the same concentration of 50 n. Furthermore, the action of TPA and DHT on PAcP secretion was blocked by five different PKC inhibitors. Results also showed that DHT, as well as TPA, could rapidly modulate PKC activity. Therefore, PKC can regulate PAcP secretion, and may also be 2001 Academic Press involved in DHT action on PAcP secretion. K: prostatic acid phosphatase; androgen regulation; protein kinase C; human prostate cancer cells. A: PAcP, prostatic acid phosphatase; ARE, androgen-responsive element; PKC, protein kinase C; PSA, prostate-specific antigen; DHT, 5-dihydrotestosterone; TPA, 12-o-tetradecanoyl phorbol-13-acetate; FBS, fetal bovine serum; PNPP, p-nitrophenyl phosphate; DMSO, dimethyl sulfoxide; Tris, Tris(hydroxymethyl)aminomethane; Hepes, N-(2-Hydroxyethyl)piperazine-N 2-ethanesulfonic acid; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; ECL, enchanced chemiluminescence; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EtBr, ethidium bromide; kb, kilobases.
INTRODUCTION Androgens play an important role in male physiology and pathology by regulating the expression of various genes in different cells (Berger and Watson, 1989; Chang et al., 1995; Swinnen et al., 1994; Swinnen et al., 1996). Androgens also regulate the secretion of several protein products (Henttu and Vihko, 1992; Lin et al., 1993a). One of *To whom correspondence should be addressed: Ming-Fong Lin, Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 984525 Nebraska Medical Center, Omaha, NE 68198-4525, U.S.A. Fax: (402) 559-6650. E-mail: [email protected] 1065–6995/01/111139+10 $35.00/0
the androgen target organs is the prostate. Thus, prostate epithelial cells serve as a classical model system for studying androgen action. Human PAcP is a prostate epithelium-specific differentiation antigen and one of the major proteins in well-differentiated prostate epithelial cells. In those cells, there are two forms of PAcP: one remains intracellular and the other is secreted (Vihko, 1979). The expression of PAcP is negligible before adolescence but a very high level of expression is observed after puberty (Reif et al., 1973). In normal adult males, both forms of PAcP are expressed. This puberty effect is the first indication 2001 Academic Press
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that the expression and secretion of PAcP may be influenced by androgens. In prostate cancer patients, the secretory form of PAcP being released into their circulation may correlate with the progression of the disease (Gutman et al., 1936). Androgen administration to these cancer patients results in an elevation of the circulating PAcP and increased cell proliferation, while the antiandrogen therapy can cause a diminished cancer cell growth and decreased PAcP level in the circulation (Blackledge et al., 1997; Huggins and Hodges, 1941; Labrie et al., 1983; Van Steenbrugge et al., 1983). Thus, for the last five decades, it has been known as a fact that androgen stimulates the secretion of PAcP. The secreted PAcP thus serves as a marker of androgen action in prostate epithelial cells and its decreased activity in the circulation serves as an indicator for the efficacy of hormonal therapy (Blackledge et al., 1997; Huggins and Hodges, 1941; Labrie et al., 1983; Van Steenbrugge et al., 1983). However, it is not known whether PAcP expression and/or secretion is a direct androgen-dependent activation, or merely an androgen-responsive process. Significant levels of PAcP have been reported in patients treated with androgen deprivation for advanced prostate cancer (Ishikawa et al., 1989; Scott et al., 1980), suggesting that, at least in this disease state, PAcP secretion may not require androgens. Androgen stimulation of PAcP secretion has been demonstrated in LNCaP cells, an androgenresponsive human prostate carcinoma cell line that expresses and secretes PAcP (Henttu and Vihko, 1992; Horoszewicz et al., 1983; Lin et al., 1993a; Schulz et al., 1985). Utilizing LNCaP cells as a model system, it can clearly be shown that androgen stimulation of PAcP secretion are at two levels (Lin et al., 1993b): the mRNA level and the secretory process. Interestingly, further studies demonstrate that in LNCaP cells, androgen could upregulate or downregulate the PAcP mRNA level, depending on the cultured cell density (Lin and Garcia-Arenas, 1994; Lin et al., 1993a; Zelivianski et al., 1998). Additionally, results from promoter studies show that the putative androgenresponsive elements (AREs) in the promoter of the PAcP gene do not respond to the androgen treatment (Shan et al., 1997). In contrast, androgen’s upregulation of the PAcP secretory pathway is independent of culture densities (Lin et al., 1993a; Lin et al., 1993b). Thus, activation of the PAcP secretory process is apparently one of the consistent events of androgen action in those cells. It is therefore important to elucidate the molecular mechanism of androgen action on the secretory
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process of PAcP in order to understand one of the signaling pathways induced by androgens in those cells. Activation of PKC signal transduction leads to alterations in the expression of a number of genes and in several enzyme activities (Cantley et al., 1991; Nishizuka, 1988; O’Brian and Ward, 1989; Rasmussen et al., 1995; Ullrich and Schlessinger, 1990). PKC is also involved in the regulation of expression and secretion of several proteins by steroid hormones (Doolan and Harvey, 1996; Kikkawa et al., 1989; Wehling, 1997). Glucocorticoid hormones, for example, antagonize PKC-stimulated secretion of ACTH (Bilezikjian et al., 1987; Mazzocchi et al., 1997; Shipston, 1995). Conversely, PKC can downregulate androgen stimulation of PSA secretion at its mRNA level (Andrews et al., 1992). However, the involvement of PKC in androgen regulation of protein secretion at the post-transcriptional level remains to be elucidated. In this communication, we investigated the mechanism that stimulates the secretion of PAcP at the post-transcriptional level in LNCaP cells. We focused our efforts on understanding the role of the PKC pathway in regulating PAcP secretion. Our results clearly indicate that PKC can effectively activate PAcP secretion and may be involved in DHT-stimulated PAcP secretion.
MATERIALS AND METHODS Materials Fetal bovine serum (FBS), RPMI-1640 medium, 4-phorbol, 12-o-tetradecanoyl phorbol-13-acetate (TPA), and the protein kinase C assay system kit were purchased from Life Technology (Grand Island, NY, U.S.A.). For androgen experiments, a certified grade FBS (Hyclone, UT, U.S.A.) which was charcoal/dextran-stripped and contained less than 74 p testosterone was used. This FBS was further heat-inactivated as described previously (Lin et al., 1993a). The steroid-reduced medium is composed of phenol red-free RPMI-1640 medium supplemented with 5% (vol/vol) certified, heatinactivated FBS (Lin et al., 1993a). Thus, the final concentration of testosterone was less than 4 p; whilst, the Kd value of this steroid to its receptor in LNCaP cells was at n levels (Horoszewicz et al., 1983). Staurosporine, A23187 calcium ionophore, H-7, calphostin C, chelerytherine, and phloretin were purchased from Calbiochem (San Diego, CA,
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U.S.A.). Hepes, Tris, PNPP, (+)-tartaric acid, citric acid and dimethyl sulfoxide (DMSO) were all from Sigma (St Louis, MO, U.S.A.). Nitrocellulose membrane was purchased from MSI (Westborough, MA, U.S.A.). The ECL reagent kit was from Amersham (Arlington Heights, IL, U.S.A.). All other reagents were obtained as described in our previous publications (Lin et al., 1992, 1993a, 1993b, 1994, 1998). Cell cultures and hormone effects The LNCaP-FGC human prostate carcinoma cell line was purchased from American Type Culture Collection and routinely maintained in RPMI-1640 medium supplemented with 5% (v/v) FBS, 1% glutamine, and 0.5% Gentamicin, as described previously (Lin et al., 1992, 1993a, 1986). LNCaP cells express endogenous PAcP, are androgenresponsive, and have been well established in studying the molecular mechanism of androgen function (Horoszewicz et al., 1983; Lin et al., 1992, 1993a, 1998). To investigate hormone effects on PAcP secretion, cells were trypsinized, seeded in the RPMI medium-5% FBS and grown for 3 days. After being grown in the steroid-reduced medium for an additional 2 days, the medium was changed to the same fresh steroid-reduced medium (Lin et al., 1993a) immediately before treatment. Various reagents including DHT, PKC activators and inhibitors that were dissolved in 100% ethanol or DMSO with 0.1% of the medium volume were added. The concentration of each reagent is specified in the figure or the figure legend. Control cells received an equal volume of solvent alone. After a period of incubation, as specified in each experiment, conditioned media were harvested and centrifuged at 4C at 1,000g for 10 min (Lin et al., 1993a). The collected supernatant was used for quantifying PAcP activity and for western blot analyses with anti-PAcP antiserum (Lin et al., 1993a, 1994, 1998). Protein concentration and acid phosphatase activity determinations The protein concentration in cell lysates was quantified using the Bio-Rad protein assay reagent with BSA as the reference (Bradford, 1976). For PAcP assay, PNPP was used as the substrate to quantify the phosphatase activity as previously described (Lin and Clinton, 1986; Lin et al., 1986). In prostate cells, the (+)-tartrate-sensitive AcP activity has been used conventionally to represent PAcP
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activity (Lin and Clinton, 1987; Lin et al., 1986). For quantifying and comparing the phosphatase activity, the assay was performed at the linear rate of reaction. Western blot analyses Conditioned media (200 l each) were electrophoresed in SDS-polyacrylamide gels (10%) and then electroblotted to nitrocellulose membranes (Lin et al., 1994, 1998). The membrane filter was treated with 5% skim milk in Tris buffered-saline containing 0.1% Tween-20 (TBST) at 24C for 1 h, followed by reaction with a rabbit anti-PAcP antiserum (Lin et al., 1994, 1998). After rinsing with TBST, the filter was incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG Ab. The PAcP protein was visualized by detecting the peroxidase activity utilizing an ECL reagent kit from Amersham (Arlington Heights, IL, U.S.A.). The relative level of PAcP protein was semiquantified by densitometric analyses of autoradiograms with Molecular Dynamics (Sunnydale, CA, U.S.A.) equipment and its software program. Northern blot analyses Total RNA was prepared from cells by a single step with Tri-reagent (Molecular Research Center, Cincinnati, OH, U.S.A.). An aliquot of each total RNA sample was electrophoresed on 1.2% agarose gels containing formaldehyde as a denaturing agent (Garcia Arenas et al., 1995; Lin and Garcia-Arenas, 1994; Lin et al., 1993a). After electrophoresis, the gel was stained with EtBr and visualized to ensure the quality of RNA and approximately equal amounts of RNA per lane, then blotted to Zeta-Probe GT membranes (Bio-Rad, CA, U.S.A.) by standard techniques (Brown and Mackey, 1997). Filters were hybridized and washed under stringent conditions as described previously (Garcia Arenas et al., 1995; Lin et al., 1993a). Both PAcP (0.29 kb) and GAPDH (0.78 kb) cDNA probes were prepared as described (Garcia Arenas et al., 1995; Lin et al., 1993a), and labeled with (-32P)dCTP using random oligonucleotide-primed synthesis (Feinberg and Vogelstein, 1983) with a commercial system from Life Technologies. For semi-quantitation, the hybridization band was visualized by autoradiography, followed by densitometric scanning of autoradiograms. Protein kinase C assay The PKC activity was quantified with The Protein Kinase C Assay System Kit from Life
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Fig. 1. (A) Dosage response of TPA effect on the secretion of PAcP. LNCaP cells were seeded in RPMI 1640 medium containing 5% FBS for 3 days. After being maintained in a steroid-reduced medium for an additional 2 days, cells were cultured in the same fresh medium. As shown in the figure, the cells were treated with different concentrations of TPA for 16 h. Control cells received the solvent alone or 50 n 4-phorbol (4-P). The conditioned media were harvested for PAcP activity assays. To quantify the PAcP protein level, conditioned media from the second set of experiments were used for Western blot analyses (Insert). The data shown was the average of duplicates and the bar indicates the range of variation. Similar results were obtained in three sets of independent experiments. (B) Time course of TPA effect on PAcP secretion. LNCaP cells were cultured as described above. Experimental cells received 50 n TPA and control cells received 50 n 4-phorbol (4-P). At different time intervals, as shown in the figure, conditioned media were harvested for PAcP activity assays. The conditioned media from the second set of experiments were used for PAcP protein analyses by Western blotting (Insert). The zero-time value for secreted PAcP activity was zero since the culture medium was replaced with fresh steroid-reduced medium immediately before the treatment with TPA. The data shown was the average of duplicates and the bar indicates the range of variation. Similar results were observed in three independent sets of experiments.
Technologies. The kinase assay was performed as described in the manufacturer’s protocol. Briefly, PKC was partially purified by a DEAE-cellulose column and its activity was quantified in the presence or absence of a specific inhibitor of PKC. The inhibitor-sensitive protein kinase activity was used to represent the specific PKC activity.
RESULTS TPA effect on PAcP secretion To examine the TPA effect on the secretion of PAcP, LNCaP cells were exposed to various concentrations of TPA for 16 h and the conditioned media were harvested for analyzing PAcP activity. As shown in Figure 1A, the secreted PAcP activity was increased by TPA treatment in a dosedependent fashion. An exposure to 10 and 50 n TPA resulted in approximately a 2- and 2.2-fold
increase in secreted PAcP activity, respectively, over the basal level of secretion. However, 50 n 4-phorbol, an inactive form of phorbol ester, did not have a significant effect on the secreted PAcP activity. At this concentration, 4-phorbol only modulated the secreted PAcP activity within a 10% range, in comparison with that in control cells that received the solvent alone (Fig. 1A). This increase in the secreted PAcP activity caused by TPA was accompanied by an increase in its protein level in conditioned media, as shown by western blot analyses (Fig. 1A, Insert). Thus, the increased PAcP activity upon TPA treatment was apparently due to an increase in its secretion and followed a dose-response pattern. To study the time-course over which TPA stimulated the secretion of PAcP, LNCaP cells were grown in the presence of 50 n TPA or 4-phorbol (50 n TPA exhibited the greatest degree of stimulation (Fig. 1A)). The secreted PAcP activity was quantified at different time intervals. Additional
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control cells received the solvent alone. As seen in Figure 1B, TPA could cause more than a 3-fold increase in the secreted PAcP activity after cells were exposed to TPA for 2–8 h, in comparison with the corresponding control that received 4-phorbol. As observed in Figure 1A, control cells that received the solvent alone secreted the same basal level of PAcP as 4-phorbol-treated cells (data not shown). Similar to the observation in Figure 1A, the increase in activity correlated with an increase in the protein level (Fig. 1B, Insert). Together, the data clearly show that TPA effectively stimulates PAcP secretion in a dose- and time-dependent fashion. To determine if the increase in secreted PAcP protein is due to an increase in its mRNA level caused by TPA, northern blot analyses were performed. Total RNA was prepared from TPAtreated cells and control cells after a 6 h exposure, since stimulation by 50 n TPA had approached its optimal effect at this time point (Fig. 1B). Northern blot analyses clearly demonstrated that TPA treatment did not cause an increase in PAcP mRNA level with a 6 h exposure although a prolonged exposure resulted in an altered mRNA expression (data not shown; Lin et al., 2000). Apparently, within this time period of treatment, TPA upregulated the secretion of PAcP at the protein level, but not at the mRNA level. Effects of PKC activator and inhibitor on TPA-stimulated PAcP secretion To delineate the molecular mechanism by which TPA stimulates PAcP secretion at the posttranscriptional level, various agents that affect the PKC pathway were tested for their effects on PAcP secretion with a treatment. H7 and staurosporine, inhibitors of several protein kinases including PKC, were tested for their effects on TPAstimulated PAcP secretion at the posttranscriptional level. Both H7 (Fig. 2A) and staurosporine (Fig. 2B) inhibited TPA action on PAcP secretion in a dose-response fashion with a 6 h exposure. To further clarify the functional role of PKC in the increase in secreted PAcP by TPA, a highly specific PKC inhibitor calphostin C was also utilized. Calphostin C significantly blocked the TPA effect at concentrations specific to the PKC inhibition (Fig. 2C). A23187 calcium ionophore, known to mobilize cellular calcium (a co-factor of PKC), exhibited a stimulatory effect on PAcP secretion (Fig. 3), similar to that caused by TPA (Fig. 1). Thus, the activation of PKC by TPA up-regulated the secretion of PAcP.
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Effect of PKC inhibitors on DHT-stimulated PAcP secretion Androgens are known classically for their upregulation of PAcP secretion (Huggins and Hodges, 1941; Lin et al., 1993a). The data shown in this communication (Figs 1, 2 & 3) clearly demonstrate that a PKC activator (TPA) is a potent stimulating factor of PAcP secretion. Thus, a possible role of PKC in DHT action on PAcP secretion was further examined utilizing PKC inhibitors. Initially, cells were exposed to 50 n DHT in the presence or absence of different dosages of calphostin C for a period of 6 h, since within this time period, DHT does not have a significant effect on the PAcP mRNA level (Lin et al., 2000). Furthermore, the concentration of 50 n DHT would exhibit an optimal effect on the secretion of PAcP by LNCaP cells since the Kd value of DHT to AREs is at n ranges (Horoszewicz et al., 1983). In the absence of calphostin C, DHT stimulated the secretion of PAcP by only approximately 10–15% over the control cells which received the solvent alone (data not shown), as reported previously (Horoszewicz et al., 1983; Lin et al., 1993a; Schulz et al., 1985). Nevertheless, calphostin C showed inhibition of the DHT effect (data not shown). To determine the significance of that inhibition, LNCaP cells were exposed to 50 n DHT for a longer period (16 h) to raise the secreted level of PAcP stimulated by DHT. As shown in Figure 4, PKC inhibitors including calphostin C (Fig. 4A), chelerytherine (Fig. 4B), and phloretin (Fig. 4C) at concentrations specifically aimed at the inhibition of PKC, blocked DHT-stimulated PAcP secretion. This inhibition of DHT-stimulated secretion of PAcP followed a dose response pattern. As controls, these reagents also inhibited TPA effects in each corresponding set of experiments (Fig. 4), similar to that observed in Figure 2. Effect of DHT on PKC activity To examine directly the functional role of PKC in DHT-stimulated PAcP secretion, we analyzed PKC activity in DHT-treated LNCaP cells, and compared it with that in TPA-treated cells. Total cellular PKC activity responded rapidly to both DHT and TPA treatments (Fig. 5). A 10 min treatment with 50 n DHT and TPA resulted in approximately 1.5- and 1.6-fold increases in PKC activity, respectively, compared to that in control cells which received the solvent alone. Within a period of 30 min, the stimulation of PKC activity by either DHT or TPA decreased to a level of
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Fig. 3. Effect of A23187 calcium ionophore on PAcP secretion. Cells were exposed to 10 n TPA, 10 n 4-phorbol (4-P), or different concentrations of A23187 for 16 h. Control cells received the solvent alone. The PAcP activity in the conditioned medium was analyzed from duplicate samples and normalized to that of the control cells.
activity, lower than the basal level in control cells (data not shown; Lin et al., 2000). Thus DHT has the same effect on PKC activity as the authentic ligand, TPA. Collectively, our data indicate that the PKC pathway is apparently involved in DHTstimulated PAcP secretion.
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It is a long-standing question as to how androgens regulate PAcP expression and secretion. Several lines of evidence indicate that the cellular form of PAcP is involved in regulating the cellular growth of prostate epithelial cells (Lin et al., 1992; Lin et al., 1998). In normal prostate epithelium, the secretion of PAcP is apparently regulated by androgens although the molecular mechanism remains unknown. In the malignant state, the elevated level of PAcP in prostate cancer patient sera
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Fig. 2. Effect of PKC inhibitors on TPA stimulation of PAcP secretion. As shown in the figure, LNCaP cells were exposed to 50 n TPA in the presence or absence of different concentrations of inhibitor H7 (A), staurosporine (B), or calphostin C (C) for 6 h. Control cells received the solvent alone or the inhibitor alone. The conditioned media were then harvested for analyzing the secreted PAcP activity. For comparison, the secreted PAcP activity was normalized to the activity of the control cells that received the solvent alone. The data shown is the average of duplicates and the bar represents the range of variation. Similar results were observed in three sets of independent experiments.
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could be due in part to an increase in the cell number of the tumor mass, increased leakage of the enzyme from depolarized plasma membranes
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Fig. 5. Effect of DHT and TPA on PKC activity. LNCaP cells were treated with 50 n DHT or TPA for different time periods as indicated. At each time point, cells were harvested, and total cellular PKC activity was assayed. The results are shown as the relative activity compared to that in control cells treated with the solvent alone. The data are the average of the activity in cells from two sets of independent experiments. The variation is less than 5%.
of carcinoma cells (Reif et al., 1973), and the prolonged half-life of the cancer-associated form of PAcP in circulation (Lin et al., 1983). Currently, androgen action on the secretion of PAcP at the molecular level still remains an enigma. Our results clearly show that the secretion of PAcP protein can be regulated by PKC in LNCaP human prostate carcinoma cells. This conclusion is supported by several lines of observation: (i) PKC activators including TPA (Fig. 1) and A23187 calcium ionophore (Fig. 3) are potent stimulators of PAcP secretion. (ii) 4-Phorbol, a biologically inactive isomer of TPA, has no significant effect on PAcP secretion (Figs 1, 2A & 3). (iii) PKC inhibitors including staurosporine, H7, calphostin C, chelerytherine, and phloretin inhibit TPAstimulated PAcP secretion (Figs 2 and 4). Alternatively, it is possible that PKC-mediated increase in PAcP secretion is in part due to its effect on cell growth. Due to the slow growth rate of LNCaP cells with a doubling time of 60–70 h in medium containing 5% FBS (Horoszewicz et al., 1983; Lin et al., 1992, 1998), there is only a Fig. 4. Effect of PKC inhibitors on PAcP secretion with DHT treatment. Cells in duplicate flasks were exposed to 50 n DHT in the presence or absence of different concentrations of PKC inhibitors: calphostin C (A), chelerytherine (B), or phloretin (C). Control cells received the solvent alone. For additional controls, in each set of experiments, cells received TPA in the presence or absence of each PKC inhibitor. After 16 h, conditioned media were harvested for analysis of the secreted PAcP activity. Similar results were observed in at least two independent sets of experiments.
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marginal growth alteration with a 6 h TPA treatment in a steroid-reduced medium. Thus, PKC plays a pivotal role in the upregulation of PAcP secretion which may represent the sum of several post-transcriptional processes including translation and/or turnover rate of the protein. Furthermore, the stimulation of PAcP secretion by TPA via the PKC pathway further indicates that the stimulated secretion of PAcP is apparently a regulatory process in LNCaP cells. Androgens raise the secreted level of PAcP by stimulating its secretory process as well as upregulating its mRNA level (Lin et al., 1993a, 1993b). Androgen action on PAcP secretion from LNCaP cells is a very slow process; e.g., a 3-day exposure to 10 n DHT results in approximately only a 2-fold elevation on the basis of equal total cellular protein (Lin et al., 1993a; Schulz et al., 1985; Horoszewicz et al., 1983). The molecular mechanism of the DHT action on PAcP secretion has not been elucidated. Although it is possible that DHT-stimulated PAcP secretion could be in part through upregulating cellular growth, our results clearly indicate that DHT rapidly activates PKC (Fig. 5), and the androgen-stimulated PAcP secretion could be blocked by a variety of PKC inhibitors with either a 6 (data not shown) or 16 h treatment (Fig. 4). Within this period of time, upregulated cell growth by DHT may not significantly contribute to its stimulation of PAcP secretion. Furthermore, the prolonged 16 h treatment with DHT results in a downregulation of PKC activity, as with the extended TPA treatment (Lin et al., 2000). Thus, the effect of androgen on PAcP secretion at the post-transcriptional level is apparently mediated in part by PKC, as with the action of other steroid hormones on protein secretion (Bilezikjian et al., 1987; Shipston, 1995). Normal prostate epithelium has two major differentiation antigens, PAcP and PSA. Although the secretion of both PAcP and PSA is known to be androgen responsive, androgen actions on secretion of these two antigens are apparently regulated by different factors involving PKC. In LNCaP cells, TPA through PKC inhibits androgen stimulation of PSA secretion rapidly, and this effect is essentially at the transcriptional level (Andrews et al., 1992). However, it is not known whether TPA action on androgen-stimulated PSA secretion could also be at the post-transcriptional level. In this communication, our results demonstrate that the PKC signal pathway could be involved in DHTstimulated PAcP secretion, and the action is at the post-transcriptional level, although a prolonged TPA treatment results in regulating the mRNA
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level (Lin et al., 2000). These differential effects on PAcP and PSA by PKC signaling may be due to the involvement of different co-factors or isoenzymes of PKC to execute PKC actions. Furthermore, it has been demonstrated that PKC exhibits differential effects in different prostate cancer cells (Henttu and Vihko, 1998). Further experiments are required to clarify these molecular mechanisms. A23187 stimulates the secretion of PAcP, similar to TPA and DHT. This suggests that androgeninduced stimulation of PAcP secretion via the PKC pathway may be due to mobilization of the cellular calcium ion, a co-factor of PKC. This is supported by the observation that DHT is involved in mobilization of cellular calcium ion (Benten et al., 1999; Lieberherr and Grosse, 1994; Steinsapir et al., 1991). Alternatively, Ca2+ may also directly activate proteases which release secretory PAcP from secretory vesicles, since it has been shown that secreted PAcP lacks the C-terminal hydrophobic peptides (Van Etten et al., 1991). This notion is supported by the observation that TPA upregulates PKC activity prior to the increased secretion of PAcP, indicating other factor(s) are involved (Figs 1B & 5). It is also puzzling that both TPA and DHT stimulated PKC activity to similar levels but the activity of secreted PAcP was much higher with TPA than with DHT. The differences between DHT and TPA stimulation of PAcP secretion could be due to different signal pathways being involved since TPA can upregulate PKC directly, while DHT may act via Ca2+ mobilization. Further experiments are required to clarify these signaling mechanisms. Nevertheless, to the best of our knowledge, this is the first report that has shed light on one novel regulatory mechanism of androgen action on PAcP secretion in prostate cancer cells. ACKNOWLEDGEMENTS This was supported in part by CA 72274, CA88184 from NCI, NIH, the Nebraska Department of Health LB595, and the BMB Prostate Cancer Research Fund. We thank Ms Renee GarciaArenas and Dr Tzu-Ching Meng for technical support, discussions and help in organizing graphs in the early phase of the studies. REFERENCES A PE, Y CY, M BT, T DJ, 1992. Tumor-promoting phorbol ester down-regulates the androgen induction of prostate-specific antigen in a human
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prostatic adenocarcinoma cell line. Cancer Res 52: 1525– 1529. B WPM, L M, S PO, W C, G Z, W F, 1999. Testosterone signaling through internalizable surface receptors in androgen receptor-free macrophages. Mol Biol Cell 10: 3113–3123. B FG, W G, 1989. Androgen-regulated gene expression. Annu Rev Physiol 51: 51–65. B LM, W JR, H T, V WW, 1987. Phorbol ester-induced down-regulation of protein kinase C abolishes vasopressin-mediated responses in rat anterior pituitary cells. Mol Endocrinol 1: 555–560. B GR, C ID, F BJ, 1997. Casodex (Bicalutamide): overview of a new antiandrogen developed for the treatment of prostate cancer. Eur Urol 31(Suppl 2): 30–39. B MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–54. B T, M K, 1997. Analysis of RNA by Northern and slot blot hybridization. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, eds. Current Protocols in Molecular Biology. New York, John Wiley & Sons. 4.9.1–4.9.16. C LC, A KR, C C, D B, G A, K R, S S, 1991. Oncogenes and signal transduction. Cell 64: 281–302. C C, S A, Y S, Y W, K E, L HJ, W C, M A, 1995. Androgen receptor: an overview. Crit Rev Eukaryot Gene Expr 5: 97–125. D CM, H BJ, 1996. Modulation of cytosolic protein kinase C and calcium ion activity by steroid hormones in rat distal colon. J Biol Chem 271: 78763–78767. F AP, V B, 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13. G A R, L FF, L D, J LP, S CC, C C, L MF, 1995. The expression of prostatic acid phosphatase is transcriptionally regulated in human prostate carcinoma cells. Mol Cell Endocrinol 111: 29–37. G EB, S EE, G AB, 1936. Significance of increased phosphatase activity of bones at the site of osteoplastic metastases secondary to carcinoma of the prostate gland. Am J Cancer 28: 485–495. H P, V P, 1998. The protein kinase C activator, phorbol ester, elicits disparate functional responses in androgen-sensitive and androgen-independent human prostatic cancer cells. Biochem Biophys Res Commun 244: 167–171. H P, V P, 1992. Steroids inversely affect the biosynthesis and secretion of human prostatic acid phosphatase and prostate-specific antigen in the LNCaP cell line. J Steroid Biochem Mol Biol 41: 349–360. H JS, L SS, K E, K JP, R H, C TM, M EA, M GP, 1983. LNCaP model of human prostatic carcinoma. Cancer Res, 43: 1809–1818. H C, H CV, 1941. Studies on prostatic cancer. I. The effect of castration, of estrogen and of androgen injection in serum acid phosphatase in metastasis carcinoma of the prostate. Cancer Res 1: 293–297. I S, S MS, V--Z R, T B, 1989. Prognostic factors in survival free of progression after androgen deprivation therapy for treatment of prostate cancer. J Urol 141: 1139–1142.
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K U, K A, N Y, 1989. The protein kinase C family: heterogeneity and its immplications. Annu Rev Biochem 58: 31–44. L F, D A, B A, L Y, R JP, H JM, G J, F AT, S J, M G, 1983. New approach in the treatment of prostate cancer: complete instead of partial withdrawal of androgens. Prostate 4: 579–594. L M, G B, 1994. Androgens increase intracellular calcium concentration and inositol 1,4,5-trisphosphate and diacylglycerol formation via a pertussis toxin-sensitive G-protein. J Biol Chem 269: 7217–7223. L MF, C GM, 1986. Human prostatic acid phosphatase has phosphotyrosyl protein phosphatase activity. Biochem J 235: 351–357. L MF, C GM, 1987. Human prostatic acid phosphatase and its phosphotyrosyl protein phosphatase activity. Adv Protein Phosphatases 4: 199–228. L MF, DV J, G-A R, 1992. Expression of human prostatic acid phosphatase activity and the growth of prostate carcinoma cells. Cancer Res 52: 4600–4607. L MF, G-A R, 1994. Effect of cell density on androgen regulation of the mRNA level of human prostatic acid phosphatase. Mol Cell Endocrinol 99: R21–R24. L MF, G-A R, C YC, L MM, P PC, X XZ, 1993a. Regulation of prostatic acid phosphatase expression and secretion by androgen in LNCaP human prostate carcinoma cells. Arch Biochem Biophys 300: 384–390. L MF, G-A R, K M, L FF, 1993b. Regulation of the expression of prostatic acid phosphatase in LNCaP human prostate carcinoma cells. Cell Mol Biol Res 39: 739–750. L MF, G-A R, X XZ, B B, L FF, 1994. The cellular level of prostatic acid phosphatase and the growth of human prostate carcinoma cells. Differentiation 57: 143–149. L MF, L CL, C GM, 1986. Tyrosyl kinase activity is inversely related to prostatic acid phosphatase activity in two human prostate carcinoma cell lines. Mol Cell Biol 6: 4753–4757. L MF, L CL, L SS, C TM, 1983. Purification and characterization of a new human prostatic acid phosphatase isoenzyme. Biochemistry 22: 1055–1062. L MF, L MS, G-A R, L FF, 2000. Differential responsiveness of prostatic acid phosphatase and prostate-specific antigen mRNA to androgen in prostate cancer cells. Cell Biol International 24: 681–689. L MF, M TC, R PS, C C, S AH, L FF, 1998. Expression of human prostatic acid phosphatase correlates with androgen-stimulated cell proliferation in prostate cancer cell lines. J Biol Chem 273: 5939–5947. M G, M LK, G G, N GG, 1997. Neuropeptide K and neurokinin A stimulate CRH and ACTH release by rat adrenal medulla in vitro. Peptides 18: 487–490. N Y, 1988. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334: 661–665. O’B CA, W NE, 1989. Biology of the protein kinase C family. Cancer Metastasis Rev 8: 199–214. R H, I CM, C R, T D, A M, G-H J, MC R, 1995. Diacylglycerol production, Ca2+ influx, and protein kinase C activation in sustained cellular reponses. Endocr Rev 16: 649–681.
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R AE, S RM, F CA, R CM, 1973. Acid phosphatase isoenzyme in cancer of the prostate. Cancer 31: 689–699. S P, B HW, F F, 1985. Steroid hormone regulation of prostatic acid phosphatase expression in cultured human prostatic carcinoma cells. Biol Chem Hoppe Seyler 366: 1033–1039. S WW, M M, W PC, 1980. Hormonal therapy of prostatic cancer. Cancer 45: 1929–1936. S JD, P K, R M, K A, L V, H P, O J, V P, 1997. Steroid-involved transcriptional regulation of human genes encoding prostatic acid phosphatase, prostate-specific antigen, and prostate-specific glandular kallikrein. Endocrinology 138: 3764–3770. S MJ, 1995. Mechanism(s) of early glucocorticoid inhibition of adrenocorticotropin secretion from anterior pituitary corticotropes. Trends Endocrinol Metab 6: 261–266. S J, S R, R P, 1991. Effects of androgen on intracellular calcium of LNCaP cells. Biochem Biophys Res Commun 179: 90–96. S JV, E M, H W, R W, V G, 1994. Androgen regulation of the messenger RNA encoding diazepam-binding inhibitor/acyl-CoAbinding protein in the human prostatic adenocarcinoma cell line LNCaP. Mol Cell Endocrinol 104: 153–162.
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S JV, V-V PP, E M, H W, V G, 1996. Androgen markedly stimulate the accumulation of neutral lipids in the human prostatic adenocarcinoma cell line LNCaP. Endocrinology 137: 4468–4474. U A, S J, 1990. Signal transduction by receptors with tyrosine kinase activity. Cell 61: 203–212. V E RL, D R, S PE, MA H, M DL, 1991. Covalent structure, disulfide bonding, and identification of reactive surface and active site residues of human prostatic acid phosphatase. J Biol Chem 266: 2313–2319. V S GJ, B MA, B V J, R JC, S FH, V P, 1983. Effect of hormone treatment on prostatic acid phosphatase in a serially transplantable human prostatic adenocarcinoma (PC-82). J Urol 129: 630–633. V P, 1979. Human prostatic acid phosphatases: purification of a minor enzyme and comparisons of the enzymes. Invest Urol 16: 349–352. W M, 1997. Specific, nongenetic actions of steroid hormones. Annu Rev Physiol 59: 365–393. Z S, C D, L MF, 1998. Cloning and analysis of the promoter activity of the human prostatic acid phosphatase gene. Biochem Biophys Res Commun 245: 108–112.
PROTEIN KINASE C PATHWAY IS INVOLVED IN REGULATING THE SECRETION OF PROSTATIC ACID PHOSPHATASE IN HUMAN PROSTATE CANCER CELLS MING-FONG LIN*†, XIU-QING ZHANG, JEANENNE DEAN and FEN-FEN LIN Department of Biochemistry/Molecular Biology, 1Section of Urologic Surgery, and 1Eppley Cancer Institute, University of Nebraska Medical Center, and 1Omaha VA Medical Center, Omaha, NE 68198, U.S.A. Received 17 January 2001; Accepted 7 June 2001 The stimulated secretion of prostatic acid phosphatase (PAcP) has been known to be a hallmark of androgen action on human prostate epithelial cells for the last five decades. The molecular mechanism of androgen action on PAcP secretion, however, has remained mostly unknown. We investigated the molecular mechanism that promotes PAcP secretion in LNCaP human prostate carcinoma cells which express PAcP and are androgen-responsive. Treatment with 12-o-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase C (PKC) activator, resulted in an increased secretion of PAcP in a dose- and time-dependent fashion. 4-Phorbol, a biologically inactive isomer of TPA, had no effect. This TPA stimulation of PAcP secretion was observed 2 h after exposure, while TPA did not have a significant effect on the mRNA level even with a 6 h treatment. A23187 calcium ionophore, known to mobilize cellular calcium which is a co-factor of PKC, also activated PAcP secretion. This TPA stimulation of PAcP secretion was more potent than the conventional stimulating agent 5-dihydrotestosterone (DHT) at the same concentration of 50 n. Furthermore, the action of TPA and DHT on PAcP secretion was blocked by five different PKC inhibitors. Results also showed that DHT, as well as TPA, could rapidly modulate PKC activity. Therefore, PKC can regulate PAcP secretion, and may also be 2001 Academic Press involved in DHT action on PAcP secretion. K: prostatic acid phosphatase; androgen regulation; protein kinase C; human prostate cancer cells. A: PAcP, prostatic acid phosphatase; ARE, androgen-responsive element; PKC, protein kinase C; PSA, prostate-specific antigen; DHT, 5-dihydrotestosterone; TPA, 12-o-tetradecanoyl phorbol-13-acetate; FBS, fetal bovine serum; PNPP, p-nitrophenyl phosphate; DMSO, dimethyl sulfoxide; Tris, Tris(hydroxymethyl)aminomethane; Hepes, N-(2-Hydroxyethyl)piperazine-N 2-ethanesulfonic acid; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; ECL, enchanced chemiluminescence; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EtBr, ethidium bromide; kb, kilobases.
INTRODUCTION Androgens play an important role in male physiology and pathology by regulating the expression of various genes in different cells (Berger and Watson, 1989; Chang et al., 1995; Swinnen et al., 1994; Swinnen et al., 1996). Androgens also regulate the secretion of several protein products (Henttu and Vihko, 1992; Lin et al., 1993a). One of *To whom correspondence should be addressed: Ming-Fong Lin, Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 984525 Nebraska Medical Center, Omaha, NE 68198-4525, U.S.A. Fax: (402) 559-6650. E-mail: [email protected] 1065–6995/01/111139+10 $35.00/0
the androgen target organs is the prostate. Thus, prostate epithelial cells serve as a classical model system for studying androgen action. Human PAcP is a prostate epithelium-specific differentiation antigen and one of the major proteins in well-differentiated prostate epithelial cells. In those cells, there are two forms of PAcP: one remains intracellular and the other is secreted (Vihko, 1979). The expression of PAcP is negligible before adolescence but a very high level of expression is observed after puberty (Reif et al., 1973). In normal adult males, both forms of PAcP are expressed. This puberty effect is the first indication 2001 Academic Press
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that the expression and secretion of PAcP may be influenced by androgens. In prostate cancer patients, the secretory form of PAcP being released into their circulation may correlate with the progression of the disease (Gutman et al., 1936). Androgen administration to these cancer patients results in an elevation of the circulating PAcP and increased cell proliferation, while the antiandrogen therapy can cause a diminished cancer cell growth and decreased PAcP level in the circulation (Blackledge et al., 1997; Huggins and Hodges, 1941; Labrie et al., 1983; Van Steenbrugge et al., 1983). Thus, for the last five decades, it has been known as a fact that androgen stimulates the secretion of PAcP. The secreted PAcP thus serves as a marker of androgen action in prostate epithelial cells and its decreased activity in the circulation serves as an indicator for the efficacy of hormonal therapy (Blackledge et al., 1997; Huggins and Hodges, 1941; Labrie et al., 1983; Van Steenbrugge et al., 1983). However, it is not known whether PAcP expression and/or secretion is a direct androgen-dependent activation, or merely an androgen-responsive process. Significant levels of PAcP have been reported in patients treated with androgen deprivation for advanced prostate cancer (Ishikawa et al., 1989; Scott et al., 1980), suggesting that, at least in this disease state, PAcP secretion may not require androgens. Androgen stimulation of PAcP secretion has been demonstrated in LNCaP cells, an androgenresponsive human prostate carcinoma cell line that expresses and secretes PAcP (Henttu and Vihko, 1992; Horoszewicz et al., 1983; Lin et al., 1993a; Schulz et al., 1985). Utilizing LNCaP cells as a model system, it can clearly be shown that androgen stimulation of PAcP secretion are at two levels (Lin et al., 1993b): the mRNA level and the secretory process. Interestingly, further studies demonstrate that in LNCaP cells, androgen could upregulate or downregulate the PAcP mRNA level, depending on the cultured cell density (Lin and Garcia-Arenas, 1994; Lin et al., 1993a; Zelivianski et al., 1998). Additionally, results from promoter studies show that the putative androgenresponsive elements (AREs) in the promoter of the PAcP gene do not respond to the androgen treatment (Shan et al., 1997). In contrast, androgen’s upregulation of the PAcP secretory pathway is independent of culture densities (Lin et al., 1993a; Lin et al., 1993b). Thus, activation of the PAcP secretory process is apparently one of the consistent events of androgen action in those cells. It is therefore important to elucidate the molecular mechanism of androgen action on the secretory
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process of PAcP in order to understand one of the signaling pathways induced by androgens in those cells. Activation of PKC signal transduction leads to alterations in the expression of a number of genes and in several enzyme activities (Cantley et al., 1991; Nishizuka, 1988; O’Brian and Ward, 1989; Rasmussen et al., 1995; Ullrich and Schlessinger, 1990). PKC is also involved in the regulation of expression and secretion of several proteins by steroid hormones (Doolan and Harvey, 1996; Kikkawa et al., 1989; Wehling, 1997). Glucocorticoid hormones, for example, antagonize PKC-stimulated secretion of ACTH (Bilezikjian et al., 1987; Mazzocchi et al., 1997; Shipston, 1995). Conversely, PKC can downregulate androgen stimulation of PSA secretion at its mRNA level (Andrews et al., 1992). However, the involvement of PKC in androgen regulation of protein secretion at the post-transcriptional level remains to be elucidated. In this communication, we investigated the mechanism that stimulates the secretion of PAcP at the post-transcriptional level in LNCaP cells. We focused our efforts on understanding the role of the PKC pathway in regulating PAcP secretion. Our results clearly indicate that PKC can effectively activate PAcP secretion and may be involved in DHT-stimulated PAcP secretion.
MATERIALS AND METHODS Materials Fetal bovine serum (FBS), RPMI-1640 medium, 4-phorbol, 12-o-tetradecanoyl phorbol-13-acetate (TPA), and the protein kinase C assay system kit were purchased from Life Technology (Grand Island, NY, U.S.A.). For androgen experiments, a certified grade FBS (Hyclone, UT, U.S.A.) which was charcoal/dextran-stripped and contained less than 74 p testosterone was used. This FBS was further heat-inactivated as described previously (Lin et al., 1993a). The steroid-reduced medium is composed of phenol red-free RPMI-1640 medium supplemented with 5% (vol/vol) certified, heatinactivated FBS (Lin et al., 1993a). Thus, the final concentration of testosterone was less than 4 p; whilst, the Kd value of this steroid to its receptor in LNCaP cells was at n levels (Horoszewicz et al., 1983). Staurosporine, A23187 calcium ionophore, H-7, calphostin C, chelerytherine, and phloretin were purchased from Calbiochem (San Diego, CA,
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U.S.A.). Hepes, Tris, PNPP, (+)-tartaric acid, citric acid and dimethyl sulfoxide (DMSO) were all from Sigma (St Louis, MO, U.S.A.). Nitrocellulose membrane was purchased from MSI (Westborough, MA, U.S.A.). The ECL reagent kit was from Amersham (Arlington Heights, IL, U.S.A.). All other reagents were obtained as described in our previous publications (Lin et al., 1992, 1993a, 1993b, 1994, 1998). Cell cultures and hormone effects The LNCaP-FGC human prostate carcinoma cell line was purchased from American Type Culture Collection and routinely maintained in RPMI-1640 medium supplemented with 5% (v/v) FBS, 1% glutamine, and 0.5% Gentamicin, as described previously (Lin et al., 1992, 1993a, 1986). LNCaP cells express endogenous PAcP, are androgenresponsive, and have been well established in studying the molecular mechanism of androgen function (Horoszewicz et al., 1983; Lin et al., 1992, 1993a, 1998). To investigate hormone effects on PAcP secretion, cells were trypsinized, seeded in the RPMI medium-5% FBS and grown for 3 days. After being grown in the steroid-reduced medium for an additional 2 days, the medium was changed to the same fresh steroid-reduced medium (Lin et al., 1993a) immediately before treatment. Various reagents including DHT, PKC activators and inhibitors that were dissolved in 100% ethanol or DMSO with 0.1% of the medium volume were added. The concentration of each reagent is specified in the figure or the figure legend. Control cells received an equal volume of solvent alone. After a period of incubation, as specified in each experiment, conditioned media were harvested and centrifuged at 4C at 1,000g for 10 min (Lin et al., 1993a). The collected supernatant was used for quantifying PAcP activity and for western blot analyses with anti-PAcP antiserum (Lin et al., 1993a, 1994, 1998). Protein concentration and acid phosphatase activity determinations The protein concentration in cell lysates was quantified using the Bio-Rad protein assay reagent with BSA as the reference (Bradford, 1976). For PAcP assay, PNPP was used as the substrate to quantify the phosphatase activity as previously described (Lin and Clinton, 1986; Lin et al., 1986). In prostate cells, the (+)-tartrate-sensitive AcP activity has been used conventionally to represent PAcP
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activity (Lin and Clinton, 1987; Lin et al., 1986). For quantifying and comparing the phosphatase activity, the assay was performed at the linear rate of reaction. Western blot analyses Conditioned media (200 l each) were electrophoresed in SDS-polyacrylamide gels (10%) and then electroblotted to nitrocellulose membranes (Lin et al., 1994, 1998). The membrane filter was treated with 5% skim milk in Tris buffered-saline containing 0.1% Tween-20 (TBST) at 24C for 1 h, followed by reaction with a rabbit anti-PAcP antiserum (Lin et al., 1994, 1998). After rinsing with TBST, the filter was incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG Ab. The PAcP protein was visualized by detecting the peroxidase activity utilizing an ECL reagent kit from Amersham (Arlington Heights, IL, U.S.A.). The relative level of PAcP protein was semiquantified by densitometric analyses of autoradiograms with Molecular Dynamics (Sunnydale, CA, U.S.A.) equipment and its software program. Northern blot analyses Total RNA was prepared from cells by a single step with Tri-reagent (Molecular Research Center, Cincinnati, OH, U.S.A.). An aliquot of each total RNA sample was electrophoresed on 1.2% agarose gels containing formaldehyde as a denaturing agent (Garcia Arenas et al., 1995; Lin and Garcia-Arenas, 1994; Lin et al., 1993a). After electrophoresis, the gel was stained with EtBr and visualized to ensure the quality of RNA and approximately equal amounts of RNA per lane, then blotted to Zeta-Probe GT membranes (Bio-Rad, CA, U.S.A.) by standard techniques (Brown and Mackey, 1997). Filters were hybridized and washed under stringent conditions as described previously (Garcia Arenas et al., 1995; Lin et al., 1993a). Both PAcP (0.29 kb) and GAPDH (0.78 kb) cDNA probes were prepared as described (Garcia Arenas et al., 1995; Lin et al., 1993a), and labeled with (-32P)dCTP using random oligonucleotide-primed synthesis (Feinberg and Vogelstein, 1983) with a commercial system from Life Technologies. For semi-quantitation, the hybridization band was visualized by autoradiography, followed by densitometric scanning of autoradiograms. Protein kinase C assay The PKC activity was quantified with The Protein Kinase C Assay System Kit from Life
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Fig. 1. (A) Dosage response of TPA effect on the secretion of PAcP. LNCaP cells were seeded in RPMI 1640 medium containing 5% FBS for 3 days. After being maintained in a steroid-reduced medium for an additional 2 days, cells were cultured in the same fresh medium. As shown in the figure, the cells were treated with different concentrations of TPA for 16 h. Control cells received the solvent alone or 50 n 4-phorbol (4-P). The conditioned media were harvested for PAcP activity assays. To quantify the PAcP protein level, conditioned media from the second set of experiments were used for Western blot analyses (Insert). The data shown was the average of duplicates and the bar indicates the range of variation. Similar results were obtained in three sets of independent experiments. (B) Time course of TPA effect on PAcP secretion. LNCaP cells were cultured as described above. Experimental cells received 50 n TPA and control cells received 50 n 4-phorbol (4-P). At different time intervals, as shown in the figure, conditioned media were harvested for PAcP activity assays. The conditioned media from the second set of experiments were used for PAcP protein analyses by Western blotting (Insert). The zero-time value for secreted PAcP activity was zero since the culture medium was replaced with fresh steroid-reduced medium immediately before the treatment with TPA. The data shown was the average of duplicates and the bar indicates the range of variation. Similar results were observed in three independent sets of experiments.
Technologies. The kinase assay was performed as described in the manufacturer’s protocol. Briefly, PKC was partially purified by a DEAE-cellulose column and its activity was quantified in the presence or absence of a specific inhibitor of PKC. The inhibitor-sensitive protein kinase activity was used to represent the specific PKC activity.
RESULTS TPA effect on PAcP secretion To examine the TPA effect on the secretion of PAcP, LNCaP cells were exposed to various concentrations of TPA for 16 h and the conditioned media were harvested for analyzing PAcP activity. As shown in Figure 1A, the secreted PAcP activity was increased by TPA treatment in a dosedependent fashion. An exposure to 10 and 50 n TPA resulted in approximately a 2- and 2.2-fold
increase in secreted PAcP activity, respectively, over the basal level of secretion. However, 50 n 4-phorbol, an inactive form of phorbol ester, did not have a significant effect on the secreted PAcP activity. At this concentration, 4-phorbol only modulated the secreted PAcP activity within a 10% range, in comparison with that in control cells that received the solvent alone (Fig. 1A). This increase in the secreted PAcP activity caused by TPA was accompanied by an increase in its protein level in conditioned media, as shown by western blot analyses (Fig. 1A, Insert). Thus, the increased PAcP activity upon TPA treatment was apparently due to an increase in its secretion and followed a dose-response pattern. To study the time-course over which TPA stimulated the secretion of PAcP, LNCaP cells were grown in the presence of 50 n TPA or 4-phorbol (50 n TPA exhibited the greatest degree of stimulation (Fig. 1A)). The secreted PAcP activity was quantified at different time intervals. Additional
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control cells received the solvent alone. As seen in Figure 1B, TPA could cause more than a 3-fold increase in the secreted PAcP activity after cells were exposed to TPA for 2–8 h, in comparison with the corresponding control that received 4-phorbol. As observed in Figure 1A, control cells that received the solvent alone secreted the same basal level of PAcP as 4-phorbol-treated cells (data not shown). Similar to the observation in Figure 1A, the increase in activity correlated with an increase in the protein level (Fig. 1B, Insert). Together, the data clearly show that TPA effectively stimulates PAcP secretion in a dose- and time-dependent fashion. To determine if the increase in secreted PAcP protein is due to an increase in its mRNA level caused by TPA, northern blot analyses were performed. Total RNA was prepared from TPAtreated cells and control cells after a 6 h exposure, since stimulation by 50 n TPA had approached its optimal effect at this time point (Fig. 1B). Northern blot analyses clearly demonstrated that TPA treatment did not cause an increase in PAcP mRNA level with a 6 h exposure although a prolonged exposure resulted in an altered mRNA expression (data not shown; Lin et al., 2000). Apparently, within this time period of treatment, TPA upregulated the secretion of PAcP at the protein level, but not at the mRNA level. Effects of PKC activator and inhibitor on TPA-stimulated PAcP secretion To delineate the molecular mechanism by which TPA stimulates PAcP secretion at the posttranscriptional level, various agents that affect the PKC pathway were tested for their effects on PAcP secretion with a treatment. H7 and staurosporine, inhibitors of several protein kinases including PKC, were tested for their effects on TPAstimulated PAcP secretion at the posttranscriptional level. Both H7 (Fig. 2A) and staurosporine (Fig. 2B) inhibited TPA action on PAcP secretion in a dose-response fashion with a 6 h exposure. To further clarify the functional role of PKC in the increase in secreted PAcP by TPA, a highly specific PKC inhibitor calphostin C was also utilized. Calphostin C significantly blocked the TPA effect at concentrations specific to the PKC inhibition (Fig. 2C). A23187 calcium ionophore, known to mobilize cellular calcium (a co-factor of PKC), exhibited a stimulatory effect on PAcP secretion (Fig. 3), similar to that caused by TPA (Fig. 1). Thus, the activation of PKC by TPA up-regulated the secretion of PAcP.
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Effect of PKC inhibitors on DHT-stimulated PAcP secretion Androgens are known classically for their upregulation of PAcP secretion (Huggins and Hodges, 1941; Lin et al., 1993a). The data shown in this communication (Figs 1, 2 & 3) clearly demonstrate that a PKC activator (TPA) is a potent stimulating factor of PAcP secretion. Thus, a possible role of PKC in DHT action on PAcP secretion was further examined utilizing PKC inhibitors. Initially, cells were exposed to 50 n DHT in the presence or absence of different dosages of calphostin C for a period of 6 h, since within this time period, DHT does not have a significant effect on the PAcP mRNA level (Lin et al., 2000). Furthermore, the concentration of 50 n DHT would exhibit an optimal effect on the secretion of PAcP by LNCaP cells since the Kd value of DHT to AREs is at n ranges (Horoszewicz et al., 1983). In the absence of calphostin C, DHT stimulated the secretion of PAcP by only approximately 10–15% over the control cells which received the solvent alone (data not shown), as reported previously (Horoszewicz et al., 1983; Lin et al., 1993a; Schulz et al., 1985). Nevertheless, calphostin C showed inhibition of the DHT effect (data not shown). To determine the significance of that inhibition, LNCaP cells were exposed to 50 n DHT for a longer period (16 h) to raise the secreted level of PAcP stimulated by DHT. As shown in Figure 4, PKC inhibitors including calphostin C (Fig. 4A), chelerytherine (Fig. 4B), and phloretin (Fig. 4C) at concentrations specifically aimed at the inhibition of PKC, blocked DHT-stimulated PAcP secretion. This inhibition of DHT-stimulated secretion of PAcP followed a dose response pattern. As controls, these reagents also inhibited TPA effects in each corresponding set of experiments (Fig. 4), similar to that observed in Figure 2. Effect of DHT on PKC activity To examine directly the functional role of PKC in DHT-stimulated PAcP secretion, we analyzed PKC activity in DHT-treated LNCaP cells, and compared it with that in TPA-treated cells. Total cellular PKC activity responded rapidly to both DHT and TPA treatments (Fig. 5). A 10 min treatment with 50 n DHT and TPA resulted in approximately 1.5- and 1.6-fold increases in PKC activity, respectively, compared to that in control cells which received the solvent alone. Within a period of 30 min, the stimulation of PKC activity by either DHT or TPA decreased to a level of
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H7 (µM)
–
–
–
50
100
100
(B) 300 Secreted PAcP activity (%)
150
200
0 TPA (nM) 4α-P (nM)
250
– – –
10 – –
– 10 –
– – 10
– – 50
– – 100
Fig. 3. Effect of A23187 calcium ionophore on PAcP secretion. Cells were exposed to 10 n TPA, 10 n 4-phorbol (4-P), or different concentrations of A23187 for 16 h. Control cells received the solvent alone. The PAcP activity in the conditioned medium was analyzed from duplicate samples and normalized to that of the control cells.
activity, lower than the basal level in control cells (data not shown; Lin et al., 2000). Thus DHT has the same effect on PKC activity as the authentic ligand, TPA. Collectively, our data indicate that the PKC pathway is apparently involved in DHTstimulated PAcP secretion.
200 150
DISCUSSION
100
It is a long-standing question as to how androgens regulate PAcP expression and secretion. Several lines of evidence indicate that the cellular form of PAcP is involved in regulating the cellular growth of prostate epithelial cells (Lin et al., 1992; Lin et al., 1998). In normal prostate epithelium, the secretion of PAcP is apparently regulated by androgens although the molecular mechanism remains unknown. In the malignant state, the elevated level of PAcP in prostate cancer patient sera
50
0 TPA (nM) Staus (nM)
– –
50 –
50 50
50 100
50 200
– 200
(C) 200 Secreted PAcP activity (%)
200 Secreted PAcP activity (%)
about 1.2-fold that of the control. The total PKC activity remained at this level for several hours. However, a prolonged exposure of 16 h to 50 n DHT or TPA resulted in a decrease in the PKC
150
100
50
0 TPA (nM) Calp (nM)
– –
50 –
50 50
50 100
50 200
– 200
Fig. 2. Effect of PKC inhibitors on TPA stimulation of PAcP secretion. As shown in the figure, LNCaP cells were exposed to 50 n TPA in the presence or absence of different concentrations of inhibitor H7 (A), staurosporine (B), or calphostin C (C) for 6 h. Control cells received the solvent alone or the inhibitor alone. The conditioned media were then harvested for analyzing the secreted PAcP activity. For comparison, the secreted PAcP activity was normalized to the activity of the control cells that received the solvent alone. The data shown is the average of duplicates and the bar represents the range of variation. Similar results were observed in three sets of independent experiments.
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(A)
Secreted PAcP activity (%)
250 200 150
50
– – –
50 – –
50 50 –
50 50 – 100 150 150 – – –
– – 50
– 150 50
(B) 250 Secreted PAcP activity (%)
DHT TPA
1.6 1.5 1.4 1.3 1.2 1.1 1.0 0
100
0 DHT (nM) Calp (nM) TPA (nM)
200 150 100 50
0 DHT (nM) Chel (µM) TPA (nM)
– – –
50 – –
50 1 –
50 5 –
– 5 –
– – 50
– 5 50
(C) 250 Secreted PAcP activity (%)
1.7 PKC activity (ratio to control)
could be due in part to an increase in the cell number of the tumor mass, increased leakage of the enzyme from depolarized plasma membranes
200 150 100 50
0 DHT (nM) Phlo (µM) TPA (nM)
– – –
50 – –
50 10 –
50 15 –
50 20 –
– 20 –
– – 50
– 20 50
20
40
60
80 100 120 140 160 180 200 Time (min)
Fig. 5. Effect of DHT and TPA on PKC activity. LNCaP cells were treated with 50 n DHT or TPA for different time periods as indicated. At each time point, cells were harvested, and total cellular PKC activity was assayed. The results are shown as the relative activity compared to that in control cells treated with the solvent alone. The data are the average of the activity in cells from two sets of independent experiments. The variation is less than 5%.
of carcinoma cells (Reif et al., 1973), and the prolonged half-life of the cancer-associated form of PAcP in circulation (Lin et al., 1983). Currently, androgen action on the secretion of PAcP at the molecular level still remains an enigma. Our results clearly show that the secretion of PAcP protein can be regulated by PKC in LNCaP human prostate carcinoma cells. This conclusion is supported by several lines of observation: (i) PKC activators including TPA (Fig. 1) and A23187 calcium ionophore (Fig. 3) are potent stimulators of PAcP secretion. (ii) 4-Phorbol, a biologically inactive isomer of TPA, has no significant effect on PAcP secretion (Figs 1, 2A & 3). (iii) PKC inhibitors including staurosporine, H7, calphostin C, chelerytherine, and phloretin inhibit TPAstimulated PAcP secretion (Figs 2 and 4). Alternatively, it is possible that PKC-mediated increase in PAcP secretion is in part due to its effect on cell growth. Due to the slow growth rate of LNCaP cells with a doubling time of 60–70 h in medium containing 5% FBS (Horoszewicz et al., 1983; Lin et al., 1992, 1998), there is only a Fig. 4. Effect of PKC inhibitors on PAcP secretion with DHT treatment. Cells in duplicate flasks were exposed to 50 n DHT in the presence or absence of different concentrations of PKC inhibitors: calphostin C (A), chelerytherine (B), or phloretin (C). Control cells received the solvent alone. For additional controls, in each set of experiments, cells received TPA in the presence or absence of each PKC inhibitor. After 16 h, conditioned media were harvested for analysis of the secreted PAcP activity. Similar results were observed in at least two independent sets of experiments.
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marginal growth alteration with a 6 h TPA treatment in a steroid-reduced medium. Thus, PKC plays a pivotal role in the upregulation of PAcP secretion which may represent the sum of several post-transcriptional processes including translation and/or turnover rate of the protein. Furthermore, the stimulation of PAcP secretion by TPA via the PKC pathway further indicates that the stimulated secretion of PAcP is apparently a regulatory process in LNCaP cells. Androgens raise the secreted level of PAcP by stimulating its secretory process as well as upregulating its mRNA level (Lin et al., 1993a, 1993b). Androgen action on PAcP secretion from LNCaP cells is a very slow process; e.g., a 3-day exposure to 10 n DHT results in approximately only a 2-fold elevation on the basis of equal total cellular protein (Lin et al., 1993a; Schulz et al., 1985; Horoszewicz et al., 1983). The molecular mechanism of the DHT action on PAcP secretion has not been elucidated. Although it is possible that DHT-stimulated PAcP secretion could be in part through upregulating cellular growth, our results clearly indicate that DHT rapidly activates PKC (Fig. 5), and the androgen-stimulated PAcP secretion could be blocked by a variety of PKC inhibitors with either a 6 (data not shown) or 16 h treatment (Fig. 4). Within this period of time, upregulated cell growth by DHT may not significantly contribute to its stimulation of PAcP secretion. Furthermore, the prolonged 16 h treatment with DHT results in a downregulation of PKC activity, as with the extended TPA treatment (Lin et al., 2000). Thus, the effect of androgen on PAcP secretion at the post-transcriptional level is apparently mediated in part by PKC, as with the action of other steroid hormones on protein secretion (Bilezikjian et al., 1987; Shipston, 1995). Normal prostate epithelium has two major differentiation antigens, PAcP and PSA. Although the secretion of both PAcP and PSA is known to be androgen responsive, androgen actions on secretion of these two antigens are apparently regulated by different factors involving PKC. In LNCaP cells, TPA through PKC inhibits androgen stimulation of PSA secretion rapidly, and this effect is essentially at the transcriptional level (Andrews et al., 1992). However, it is not known whether TPA action on androgen-stimulated PSA secretion could also be at the post-transcriptional level. In this communication, our results demonstrate that the PKC signal pathway could be involved in DHTstimulated PAcP secretion, and the action is at the post-transcriptional level, although a prolonged TPA treatment results in regulating the mRNA
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level (Lin et al., 2000). These differential effects on PAcP and PSA by PKC signaling may be due to the involvement of different co-factors or isoenzymes of PKC to execute PKC actions. Furthermore, it has been demonstrated that PKC exhibits differential effects in different prostate cancer cells (Henttu and Vihko, 1998). Further experiments are required to clarify these molecular mechanisms. A23187 stimulates the secretion of PAcP, similar to TPA and DHT. This suggests that androgeninduced stimulation of PAcP secretion via the PKC pathway may be due to mobilization of the cellular calcium ion, a co-factor of PKC. This is supported by the observation that DHT is involved in mobilization of cellular calcium ion (Benten et al., 1999; Lieberherr and Grosse, 1994; Steinsapir et al., 1991). Alternatively, Ca2+ may also directly activate proteases which release secretory PAcP from secretory vesicles, since it has been shown that secreted PAcP lacks the C-terminal hydrophobic peptides (Van Etten et al., 1991). This notion is supported by the observation that TPA upregulates PKC activity prior to the increased secretion of PAcP, indicating other factor(s) are involved (Figs 1B & 5). It is also puzzling that both TPA and DHT stimulated PKC activity to similar levels but the activity of secreted PAcP was much higher with TPA than with DHT. The differences between DHT and TPA stimulation of PAcP secretion could be due to different signal pathways being involved since TPA can upregulate PKC directly, while DHT may act via Ca2+ mobilization. Further experiments are required to clarify these signaling mechanisms. Nevertheless, to the best of our knowledge, this is the first report that has shed light on one novel regulatory mechanism of androgen action on PAcP secretion in prostate cancer cells. ACKNOWLEDGEMENTS This was supported in part by CA 72274, CA88184 from NCI, NIH, the Nebraska Department of Health LB595, and the BMB Prostate Cancer Research Fund. We thank Ms Renee GarciaArenas and Dr Tzu-Ching Meng for technical support, discussions and help in organizing graphs in the early phase of the studies. REFERENCES A PE, Y CY, M BT, T DJ, 1992. Tumor-promoting phorbol ester down-regulates the androgen induction of prostate-specific antigen in a human
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