The Conventional Transforming Growth Factor-β (TGF-β) Receptor Type I Is Not Required for TGF-β1 Signaling in a Human Prostate Cancer Cell Line, LNCaP

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

241, 151–160 (1998)

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The Conventional Transforming Growth Factor-b (TGF-b ) Receptor Type I Is Not Required for TGF-b1 Signaling in a Human Prostate Cancer Cell Line, LNCaP1 Isaac Yi Kim,2 David J. Zelner, and Chung Lee3 Department of Urology, Northwestern University Medical School, Chicago, Illinois 60611

LNCaP is an androgen-responsive human prostate cancer cell line that has a defective gene for ALK-5, the conventional TGF-b receptor type I. Yet, these cells respond to exogenous TGF-b1 under appropriate concentrations of dihydrotestosterone (DHT). Because a heteromeric complex composed of type I and type II receptor is required for TGF-b signaling, the expression of these receptors was investigated in LNCaP cells at following concentrations of DHT — 0, 0.1, and 100 nM. These concentrations were selected because they represent the zero DHT control in which LNCaP cells are not sensitive to TGF-b1, the proliferative dose of DHT in which these cells are sensitive to exogenous TGF-b1, and the growth-arrest dose of DHT in which LNCaP exhibits signs of TGF-b signaling but are insensitive to exogenous TGF-b1, respectively. Results of Western blot analysis showed that LNCaP cells express an increased level of type II receptor at 0.1 nM DHT, the TGF-b1-sensitive dose. However, results of competitive quantitative RT-PCR demonstrated that DHT did not significantly change the level of type II receptor mRNA, suggesting that DHT modulates the level of type II receptor at the posttranscriptional level. In contrast, ALK-5 was not detected in these cells by either Western blot analysis or RT-PCR at all concentrations of DHT used in this study. Subsequently, the expression of ALK-1, -2, and -4 in LNCaP cells was examined because these proteins have been shown to bind TGF-b1 in vitro. ALK1 and -2 were detected in these cells. Further analysis by competitive quantitative RT-PCR and Western blot demonstrated that DHT did not affect the level of expression of ALK-1 and -2 in LNCaP cells. These observations, taken together, demonstrate that ALK-5 is not required for TGF-b1 signaling and that there may 1 This work was supported in part by NIH Grants DK 39250 and CA 60553. 2 Present address: Scott Department of Urology, Baylor College of Medicine, 6535 Fannin, F-403, Houston, TX 77030. 3 To whom reprint requests should be addressed at Tarry 11-715, Department of Urology, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 60611.

be alternative mechanism(s) for TGF-b1 signal transduction in some systems. q 1998 Academic Press Key Words: TGF-b; TGF-b receptors; TGF-b signaling; prostate cancer.

INTRODUCTION

Transforming growth factor-b (TGF-b ) is a multifunctional growth factor expressed by many cell lines and tissue types. Although this growth factor has been implicated in immunosuppression, angiogenesis, chondrogenesis, myogenesis, epithelial–mesenchymal interaction, and production of extracellular matrix protein, it usually acts as a potent growth inhibitor in most cells, especially those of the epithelial lineage [reviewed in 1–3]. TGF-b exerts its effects through an interaction with membrane receptors. There are three ubiquitously expressed TGF-b receptors—type I, II, and III (TbR-I, TbR-II, and TbR-III, respectively). Molecular weights of TbR-I, TbR-II, and TbR-III are 50– 55, 65–75, and 200–300 kDa, respectively [4]. TbRIII is a membrane proteoglycan that has a very short cytoplasmic tail and lacks any consensus signaling motif [5], whereas TbR-II and TbR-I are serine/threonine kinases [6–8]. The current evidence concerning the mechanism of action of TGF-b receptors suggests that both TbR-I and TbR-II are required for TGF-b signal transduction [9]. Wrana et al. have proposed a model for the mechanism of action of TGF-b receptors in which TbR-II functions as a constitutive kinase and activates TbR-I which, in turn, initiates the TGF-b signal transduction cascade [10]. According to this model, TbR-I requires TbR-II for ligand binding and TbR-II requires TbR-I for signal transduction. To date, only one each of TbR-II and TbR-I (ALK-5 or R4) has been identified and characterized [6–8]. In addition, three additional proteins with molecular weight similar to that of TbR-I have been shown to bind TGF-b1 in vitro: ALK-1 (TSR-1), ALK-2 (Tsk7L or ActR-I), and ALK-4 (ActR-IB) [11–14]. However, ALK-1, -2, and -4 are not

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0014-4827/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.

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considered to be a functional TbR-I at the present time because in mink lung epithelial cells that lack TbR-I, sensitivity to TGF-b1 was restored when these cells were transfected with ALK-5 but not ALK-1, -2, and -4 [7, 8, 11–14]. Therefore, ALK-5 is designated as the conventional TbR-I. LNCaP is an androgen-responsive human prostate cancer cell line that was originally derived from a lymph node metastasis [15]. These cells have a mutated androgen receptor at the androgen binding domain and respond not only to androgens but also to antiandrogens, estrogen, and progestins [16, 17]. LNCaP cells exhibit a bell-shaped growth response to androgen stimulation [18, 19]. At low concentrations of dihydrotestosterone (DHT) (0.001 to 0.1 nM), these cells proliferate in a dose-dependent manner. When DHT concentrations in the medium exceed 1.0 nM, the rate of proliferation declines in a dose-dependent manner. Therefore, DHT concentrations lower than 0.1 nM are considered as the proliferative doses while those greater than 1.0 nM are considered as the growth-arrest doses. While the mechanism of androgen action in LNCaP proliferation and growth arrest remains undefined, TGF-b signaling has been implicated as one of the mediators of the androgen-regulated growth arrest [20]. Although LNCaP cultures are normally not sensitive to exogenous TGF-b1, they respond to the inhibitory effect of TGF-b1 in cultures that contain the proliferative doses of DHT [21] as well as epidermal growth factor (EGF) and transforming growth factor-a (TGFa) [22]. In addition, when the culture medium contains the growth-arrest dose of DHT, TGF-b1 signaling can be manifested in LNCaP because these cells synthesize and secrete a high level of TGF-b1 under this condition [20]. Regarding TGF-b receptors, LNCaP cells express TbR-II [21] but not the conventional TbR-I because these cells have a defective ALK-5 gene resulting in an undetectable level of ALK-5 mRNA and protein [23]. These series of observations suggest that LNCaP cells may respond to TGF-b1 in the absence of the conventional TbR-I. Therefore, the present study has investigated the sensitivity to TGF-b1 and the expression of TbR-I and TbR-II in LNCaP cells under varying concentrations of DHT. We report that TGF-b1 signaling occurs in these cells in the apparent absence of the conventional TbR-I, ALK-5. EXPERIMENTAL PROCEDURES Cell culture. LNCaP and PC3 cells were purchased from the American Type Culture Collection (Rockville, MD). In this study, all cells were derived from the 28th through 31st passages. Cells were routinely maintained in RPMI 1640 containing 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 mg/ml). Phenol red-free RPMI 1640 supplemented with dextran-treated charcoal-stripped serum (cFBS) was used in each experiment. Crystalline

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DHT (Sigma Chemical Co., St. Louis, MO) was dissolved in absolute ethanol at a concentration of 1003 M and was reconstituted in the culture medium at concentrations ranging from 100 to 0.001 nM. Human TGF-b1 (Collaborative Research, Bedford, MA) was diluted to 2 mg/ml and added to the culture medium at preselected concentrations. Mitogenic assay. LNCaP cells at 1 1 104/well were plated in 24well culture plates in RPMI 1640 supplemented with 10% FBS and allowed to adhere for 24 h. The following day, designated as day 0, cultures were washed twice with phosphate-buffered saline (PBS) and cells from randomly selected wells were harvested and counted to determine the plating efficiency. Cells in the remaining wells were cultured for 4 days in phenol red-free RPMI 1640 supplemented with cFBS containing TGF-b1 and different concentrations of DHT. Media were changed once on day 2. To count cells, media were removed and cells were detached with 0.5 ml of 0.05% trypsin, added to 19.5 ml of isotonic solution (Isoton II, Coulter Corp., Hialeah, FL), and counted with a Coulter counter (ZF, Coulter Corp.). Triplicate wells were used for each experiment. All mitogenic assays were performed at least three times and similar results were obtained each time. Transient transfection and luciferase activity assay. Cells were seeded in six-well plates at 1 1 105 cells/well. Transient transfection experiments were carried out using lipofectamine according to the protocol recommended by the manufacturer (Gibco-BRL, Grand Island, NY). The plasmid, p3TP-Lux, contains three TPA-responsive elements from the human collagenase gene and one TGF-b-responsive element from the human plasminogen activator inhibitor-1 (PAI1) promoter linked to the luciferase reporter gene [9]. One microgram per milliliter of p3TP-Lux along with or without 1 mg/ml TGF-b receptors (TbR-I or TbR-II) cDNA [6, 7] and 12 ml/ml lipofectamine were added to each well and incubated for 24 h. Finally, 5 ng/ml TGF-b1 was added and cultures were maintained for additional 16 h according to the original protocol [9]. The extent of promoter activity of p3TP-Lux was assayed by measuring luciferase activity using a commercial luciferase assay kit (Enhanced luciferase assay kit 1800k, Analytical Luminescence Laboratory). All assays were performed at least three times and similar results were obtained each time. Western blot analysis. Cells were harvested by scraping and homogenized in PBS at 47C. Protein concentrations were determined and samples were placed in sample buffer (0.0625 M Trizma base, 2% SDS, 5% 2-mercaptoethanol) and boiled for 5 min. Electrophoresis was performed in 10% SDS–polyacrylamide gel using 100 mg of total protein loaded onto each lane. Following electrophoresis, proteins were transferred to a 0.2 mm nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was incubated overnight in blocking buffer (3% bovine serum albumin, 0.05% Tween, 20 mM Tris–Cl, pH 7.2, and 500 mM NaCl). Subsequently, the membrane was incubated with the appropriate antibody (anti-TbR-II, -ALK-1, -ALK-2, -ALK-4, or -ALK-5) [7, 11] at 47C overnight. After washing with 0.1% PBS–Tween, the membrane was incubated in the presence of goat anti-rabbit alkaline phosphatase-labeled secondary antibody (BioRad). After washing few times, the immunoreactive proteins were visualized by enhanced chemiluminescence (Amersham Corp., Arlington Heights, IL). Immunoprecipitation. Cells were harvested by scraping and lysed in buffer (25 mM Tris, 50 mM NaCl, 0.3 mM PMSF) for 30 min on ice. Immunoprecipitation was carried out with the cell lysates using anti-TbR-II or anti-TbR-I antibodies [7]. Briefly, antibodies were added to cell lysates and incubated on ice for 1 h. Then, protein A–Sepharose was mixed with the antibody/antigen lysate and incubated for additional 1 h at 47C. Subsequently, the mixture was washed three times and resuspended in SDS gel loading buffer (50 mM Tris–Cl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol). Samples were subjected to Western blot analysis using the same antibodies as previously described.

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CONVENTIONAL TGF-b RECEPTOR TYPE I AND TGF-b1 SIGNALING Poly(A) RNA isolation and reverse transcriptase–polymerase chain reaction (RT-PCR). Cells were harvested and immediately frozen in liquid nitrogen. Subsequently, they were processed for poly(A) RNA isolation using Mini Ribosep Ultra mRNA isolation kit according to the protocol recommended by the manufacturer (Collaborative Medical Products, Bedford, MA). RT-PCR was performed using the Gene AMP RNA PCR kit (Perkin-Elmer, Norwalk, CT) with 200 to 400 ng of poly(A) RNA. The following primers were used. TbR-II: 5* primer, 5* AGCAG AAGCT GAGTT CAACC TGGC3 * (nucleotides 580–604); 3 * primer, 5*GGAGC CATGT ATCTT GCAGT TCCC3* (nucleotides 1256–1281). ALK-1: 5* primer, 5*GAGTC CAGTC TCATC CTGAA AGC3* (nucleotides 566–588); 3* primer, 5*CTCTT GACCA GCACA TTGCG3* (nucleotides 1091–1110). ALK-2: 5* primer, 5*CGTGA TGAGA AGTCA TGGTT CAGGG3* (nucleotides 821–845); 3 * primer, 5*TACGC AGTGC TGTGA GTCTT GCG3* (nucleotides 1552–1574). ALK-4: 5* primer, 5*GAG CAC GGG TCC CTG TTT GA3* (nucleotides 856– 876); 3* primer, 5*TCG CAT CAT CTT CCC CAT CAC3 * (nucleotides 1402–1423). ALK-5 primer set 1: 5* primer, 5*TTGCT GGACC AGTGT GCTTC G3 * (nucleotides 388–408); 3 * primer, 5*CCATC TGTTT GGGAT ATTTG GCC3 * (nucleotides 1352–1374). ALK-5 primer set 2: 5* primer, 5*GCGAC GGCGT TACAG TGTTT CTGC3* (nucleotides 91–114); 3 * primer, 5*ATGGT GAATG ACAGT GCGGT TGTGG3* (nucleotides 443–468). The conditions for RT-PCR were identical for all six primer sets. Reverse transcription was performed using the 3* primers at the following conditions: 457C for 15 min, 997C for 5 min, and 57C for 5 min. After reverse transcription, PCR was performed as follows: 947C for 1 min, 557C for 1 min, and 727C for 1.5 min for 35 cycles followed by a 10-min incubation at 727C. To visualize the PCR products, the samples were subjected to electrophoresis in 1% agarose gel followed by staining with ethidium bromide. The authenticity of the products were confirmed by diagnostic restriction digests with the following enzymes: TbR-II, BstXI; ALK1, XhoI; ALK-2, XbaI; ALK-4, PvuII; ALK-5 product 1, BamHI; ALK5 product 2, XhoI. For quantitation of mRNA, competitive quantitative RT-PCR was performed [24]. To generate the competitors to serve as the internal standards, the previously described low-stringency PCR method was used [25]. Briefly, RT-PCR was carried out at a lower annealing temperature (457C) to decrease the stringency of priming. This yielded multiple products because lower stringency allows more mismatches in primer sequences. As competitors, fragments that were easily separable from the target by electrophoresis on an agarose gel were selected. Because the competitors were generated using the same primers as those of the target, the sequence of the priming sites is identical to that of native target message. Once appropriate competitors were generated, they were added at 80 fg to each RTPCR. The resulting products were visualized as previously described. Quantitation of the resulting bands was carried out by means of an automated image analysis system (PDQUEST, Pdi, Huntington Station, NY). The relative integrated density of each band was calculated by taking the absorbance multiplied by the surface area. The ODs were subsequently converted to ratios between the target and the competitor. Statistical analysis. All numerical data are expressed as means { SEM (standard error of the mean) with triplicate observations. Differences of means among different treatments were compared by unpaired Student’s t test [26]. A value of P õ 0.05 was considered statistically significant.

RESULTS

TGF-b1 sensitivity in LNCaP cells. The effect of TGF-b1 on LNCaP proliferation was assessed by changes in cell count between TGF-b1-treated and un-

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treated cultures. Figure 1A shows LNCaP cell count as the percent of the control culture after 4 days of treatment with increasing doses of TGF-b1 in the presence of varying concentrations of DHT. A significant decrease in cell count following TGF-b1 treatment was observed only when cells were treated with 0.1 nM DHT. TGF-b1 was effective at 1 ng/ml or greater. At 10 and 100 ng/ml of TGF-b1, the cell count decreased to Ç45% of that of the control. In addition to an inhibition in cellular proliferation, activation of a TGF-b-responsive promoter present in the plasmid, p3TP-Lux, is also a distinct effect of TGFb1 for the following reason. In p3TP-Lux, the luciferase reporter gene is under the control of a TGF-b-responsive promoter composed of three TPA-responsive elements from the human collagenase gene and one TGFb-responsive element from the human PAI-1 gene [9]. To provide further evidence that LNCaP are sensitive to TGF-b1, cells were transiently transfected with p3TP-Lux and luciferase activity was assayed as an extent of TGF-b1 sensitivity. Figure 1B shows that a treatment with 5 ng/ml of TGF-b1 resulted in a significant increase in luciferase activity only when LNCaP cells were cultured in the presence of 0.1 nM DHT. The levels of luciferase activity in cultures that were not treated with TGF-b1 were used as respective controls. These observations are consistent with that of the previous study which reported that LNCaP cells were sensitive to TGF-b1 when the culture contained 0.1 nM DHT [21]. TbR-II expression in LNCaP cells. Because TGF-b1 signaling requires a heteromeric complex composed of TbR-II and TbR-I [9], the expression of these receptors was investigated by Western blot analysis and RTPCR. To enhance the sensitivity of detection for TbRII protein, Western blot analysis was carried out following immunoprecipitation. Figure 2A shows a dose-dependent increase in the level of TbR-II protein at DHT concentrations between 0 and 0.1 nM. At 100 nM DHT, the level of TbR-II protein decreased dramatically. To quantitate the changes in TbR-II mRNA following treatment with different levels of DHT, competitive quantitative RT-PCR [24] was performed because Northern blot analysis could not detect the message (Fig. 2B). The authenticity of the product was confirmed by a diagnostic restriction digest with BstXI. As an internal standard for quantitation, a competitor was generated using the previously described low-stringency method [25]. RT-PCR performed at a lower stringent condition (annealing temperature of 457C) yielded multiple bands. Of these, we chose an Ç450-base product as the competitor because it was easily separable from the native TbR-II message by electrophoresis using a 1% agarose gel. This competitor can be used as

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FIG. 1. TGF-b1 sensitivity in LNCaP cells. (A) Effect of increasing doses of TGF-b1 on cell numbers. Cells were seeded at 1 1 104/well in 24-well plates and cultured in medium containing TGF-b1 at 0, 0.1, 1, 10, and 100 ng/ml and following concentrations of DHT: zero DHT control, 0.001, 0.1, and 100 nM. Cell were counted at the end of 4 days by Coulter counter. Media were changed once on day 2. The results are presented as a percentage of control. (B) Effect of TGF-b1 on gene transcriptional activation of p3TP-Lux. Cells were seeded at 1 1 105/well in 6-well plates and cultured for 2 days with following concentrations of DHT: zero DHT control, 0.001, 0.1, and 100 nM. Subsequently, cells were transfected transiently with p3TP-Lux by lipofection. At 48 h after the transfection, cells were incubated with 5 ng/ml of TGF-b1 for 16 h. Luciferase activity was measured and expressed as a percentage of control. Each value represents the mean of triplicate observations. The vertical bar denotes standard error of the mean. *P õ 0.05 when compared with the corresponding value in cultures grown in the absence of TGF-b1.

a standard to quantitate the amount of TbR-II mRNA because the new product contains the identical primer sequence as the native TbR-II mRNA. For quantitation, 80 fg of the competitor was added to 200 ng each of poly(A) RNA sample for RT-PCR reaction. The products were visualized by electrophoresis using 1% agarose gel followed by staining with ethidium bromide. The optical density of each band was obtained and converted to a ratio between the target and the competitor (data not shown). The results demonstrated that the level of TbR-II mRNA in LNCaP cells did not change significantly with increasing doses of DHT. These results indicated that DHT regulated the expression of TbR-II in LNCaP cells at the posttranscriptional level. TbR-I expression in LNCaP cells. Unlike TbR-II, the conventional TbR-I (ALK-5) was not detected in LNCaP cells. Specifically, results of Western blot analysis for the receptor showed that the expected Ç55kDa protein was observed in the positive control PC3 cells but not in LNCaP cells at all the concentrations of DHT investigated in this study (Fig. 3A). To enhance the probability of detecting ALK-5 protein, Western blot analysis was also carried out following immuno-

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precipitation. However, ALK-5 could not be successfully separated from the IgG fraction (data not shown). To investigate the possibility that the undetectable level of ALK-5 protein in LNCaP cells is due to a low level of the respective mRNA, RT-PCR was performed. The results showed that RT-PCR using 400 ng of poly(A) RNA could not detect ALK-5 mRNA in LNCaP cells at all concentrations of DHT used in this study (Fig. 3B, top). The expected 986-base product was detected in the RNA fraction isolated from PC3 cells, indicating that the lack of RT-PCR product in LNCaP cells was not due to defective primers. RT-PCR performed in the absence of reverse transcriptase yielded no product, demonstrating the absence of a significant amount of genomic DNA contamination. The inability to detect ALK-5 mRNA in LNCaP cells was not likely due to a RNA degradation because the same RNA samples yielded positive product when TbR-II-specific primers were used (Fig. 3B, bottom). The authenticity of the products was confirmed by diagnostic restriction digests with the following enzymes: TbR-I, BamHI; TbRII, BstXI (data not shown). To further support the above RT-PCR result, a sec-

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FIG. 2. Expression of TbR-II in LNCaP cells. (A) Western blot analysis. Cells were cultured in varying concentrations of DHT for 4 days. Media were changed once on day 2. Subsequently, cells were harvested, lysed, and immunoprecipitated using anti-TbR-II antibody. Then, samples were separated by electrophoresis in 10% SDS–polyacrylamide gel and immunoreactive bands were visualized by enhanced chemiluminescence. PC3 cells were used as a positive control. (B) Competitive quantitative RT-PCR. For each analysis, 200 ng of poly(A) RNA was used. The competitor was generated by the previously described low-stringency method. For each reaction, 80 fg of the competitor was used. Products were separated by electrophoresis using 1% agarose gel and visualized by staining with ethidium bromide. PC3 cells were used as a positive control. To control for genomic DNA contamination, all reactions were also performed in the absence of reverse transcriptase. The highest level of TbR-II protein was observed at 0.1 nM DHT, the TGF-b1-sensitive dose. However, no significant change in the level of TbR-II mRNA occurred with varying concentrations of DHT, indicating that DHT regulates the expression of TbR-II at the posttranscriptional level. (RT) Reverse transcriptase.

ond set of ALK-5 primers was generated. This new set of primers was designed against the extracellular domain because the first set of primers was targeted to the intracellular domain of the receptor molecule. As with the original RT-PCR result, no product was detected in LNCaP cells (Fig. 3B, middle). The lack of a product was again not due to defective primers as the expected 377-base product was observed in the positive control cell line, PC3. The absence of the RT-PCR product was not due to a RNA degradation because TbR-II primers yielded positive product in all RNA samples. The authenticity of the product was confirmed by a diagnostic restriction digest with XhoI (data not shown). An investigation of the sensitivity of the primers demonstrated that both sets of primers require at the least Ç3 1 105 copies of ALK-5 cDNA for a positive detection by ethidium bromide staining in agarose gels (data not shown). In the standard poly(A) RNA isolation used in this study, the average yield was Ç20 mg/ 8.0 1 107 cells. Then, an aliquot of 400 ng of poly(A) RNA used in each RT-PCR is equivalent to using Ç1.6 1 106 cells. Assuming that the efficiency of reverse transcription is comparable to that of oligo(dT) primers (Ç40%) [27], the results of RT-PCR indicated that LNCaP cells express ALK-5 mRNA at a level lower than one copy for every 2.1 cells at the DHT dose of 0.1 nM which renders LNCaP cells sensitive to exogenous TGF-b1.

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Expression of the three putative TbR-Is in LNCaP cells. Above results indicated that LNCaP cells respond to TGF-b1 in the apparent absence of the conventional TbR-I. Because a heteromeric complex composed of TbR-I and TbR-II is required for TGF-b signaling, the expression of ALK-1, -2, and -4 in LNCaP cells was investigated. Previously, these proteins have been shown to bind TGF-b1 in vitro [11–14]. Figure 4 shows the result of RT-PCR for these receptors. LNCaP cells expressed ALK-1 and -2. However, ALK-4 was not detected in these cells regardless of the concentration of DHT present in the culture medium (data not shown). Authenticity of the products was confirmed by diagnostic restriction digests with the following enzymes: ALK1, XhoI; ALK-2, XbaI; ALK-4, PvuII. Control reactions for a possible genomic DNA contamination yielded no product. As an initial attempt to identify the functional TbRI in LNCaP cells, the effect of DHT on the level of expression of ALK-1 and -2 in these cells was investigated by Western blot analysis (Fig. 5A) and competitive quantitative RT-PCR (Fig. 5B). The hypothesis was that the level of expression of the functional TbRI may also increase in a manner similar to that of TbRII at the dose of DHT (0.1 nM) which rendered LNCaP cells sensitive to TGF-b1. The competitor for quantitative RT-PCR was generated as previously described using the low-stringency method [25]. Products were vis-

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FIG. 3. Expression of the conventional TbR-I in LNCaP cells. (A) Western blot analysis. Cells were cultured in varying concentrations of DHT for 4 days. Media were changed once on day 2. Subsequently, cells were harvested and lysed using protein sample buffer. Then, 100 mg of each protein sample was separated by electrophoresis in 10% SDS–polyacrylamide gel and immunoreactive bands were visualized by enhanced chemiluminescence. PC3 cells were used as a positive control. (B) RT-PCR analysis. For each analysis, 400 ng of poly(A) RNA was used. Products were separated by electrophoresis using 1% agarose gel and visualized by staining with ethidium bromide. PC3 cells were used as a positive control. To control for genomic DNA contamination, all reactions were also carried out in the absence of reverse transcriptase. TbR-I was not detected in LNCaP cells at all concentrations of DHT investigated in this study. The absence of TbR-I RTPCR product in LNCaP cells was not likely due to a RNA degradation because the same RNA sample yielded product for TbR-II. (RT) Reverse transcriptase.

ualized as previously described. The optical density of each band was determined and converted to a ratio between the target and the competitor (data not shown). The results demonstrated that DHT did not significantly alter the levels of expression of these two putative TbR-Is at both the protein and the mRNA level in LNCaP cells. DISCUSSION

Results of the present study demonstrated that LNCaP cells responded to TGF-b1 in the apparent absence of ALK-5, the conventional TbR-I. Specifically, TGF-b1 inhibited cellular proliferation and activated a TGF-b-responsive promoter when these cells were treated with 0.1 nM DHT. By Western blot analysis, it was shown that these cells have an elevated level of TbR-II protein at 0.1 nM DHT. ALK-5, on the other hand, was not detected in LNCaP cells by Western blot

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analysis and RT-PCR at all concentrations of DHT investigated in this study. However, the two putative TbR-Is (ALK-1 and -2) were detected in these cells at all concentrations of DHT used in this study. These observations, taken together, demonstrate for the first time that TGF-b signaling does not require ALK-5 and suggest that there may be additional mechanism(s) for TGF-b signaling in some systems. LNCaP is an androgen-responsive human prostate cancer cell line that exhibits a bell-shaped growth response to androgen stimulation. Recently, DHT was shown to modulate TGF-b1 sensitivity in these cells [21]. Results of the present study are consistent with these findings in that TGF-b1 inhibited cellular proliferation and activated a TGF-b-responsive promoter in LNCaP cells only when the medium contained 0.1 nM DHT. An investigation of the expression of TGF-b receptors showed that the level of TbR-II protein increased dramatically in these cells only at 0.1 nM DHT.

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FIG. 4. Expression of putative TbR-Is in LNCaP cells. For each RT-PCR analysis, 400 ng of poly(A) RNA was used. Products were separated by electrophoresis using 1% agarose gel and visualized by staining with ethidium bromide. PC3 cells were used as a positive control. To control for genomic DNA contamination, all reactions were also performed in the absence of reverse transcriptase. ALK-1 and -2 were detected in LNCaP cells at all concentrations of DHT investigated in this study. (RT) Reverse transcriptase.

Since the level of TbR-II mRNA did not significantly change in response to DHT treatment, androgen likely regulates the expression of TbR-II at the posttranscriptional level. In the absence of DHT, LNCaP was not sensitive to TGF-b1 and the level of TbR-II protein was undetectable. Therefore, when LNCaP cells responded to TGF-b1, the level of TbR-II was relatively high. It should be noted, though, that a substantial level of TbR-II was present in these cells at 0.001 nM DHT. Since LNCaP cells are insensitive to TGF-b1 at this particular concentration of DHT, it is likely that the amount of TbR-II expressed under this condition is not sufficient for TGF-b signaling to occur. Alternatively, there may be additional TGF-b signaling molecules that are expressed at 0.1 nM and not at 0.001 nM DHT. Further experiments are necessary to verify this concept. Current understanding regarding the mechanism of action of TGF-b receptors states that a heteromeric complex composed of TbR-I and TbR-II is required for TGF-b signaling. Wrana et al. have proposed that there is an initial binding between the growth factor and TbR-II which in turn recruits TbR-I and initiates the signal transduction cascade [10]. Such mechanism of receptor activation appears to be a consistent theme among the members of TGF-b superfamily [28–30]. At present, one each of TbR-II and TbR-I (ALK-5 or R4) has been cloned and characterized [6–8]. Three additional proteins that bind TGF-b1 in vitro and have molecular weight similar to that of TbR-I have been iden-

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tified: ALK-1 (TSR-1), ALK-2 (Tsk7L or ActR-I), and ALK-4 (ActR-IB) [11–14]. At present, only ALK-5 is considered to be a functional TbR-I because ALK-5, but not ALK-1, -2, and -4, has been demonstrated to restore TGF-b1 sensitivity in TbR-I deficient mink lung epithelial cells [7, 8, 11–14]. As a result, ALK-5 is designated as the conventional TbR-I. Recently, ten Dijke et al. have demonstrated that ALK-2 and -4 are functional activin receptor type I in porcine aortic endothelial cells [11]. Nevertheless, the possibility remains that ALK1, -2, and -4 and other yet unidentified proteins may serve as the functional TbR-Is in systems other than mink lung epithelial cells and porcine aortic endothelial cells. In this regard, a recent publication has reported a mammary cell line that responds to TGF-b1 in the apparent absence of ALK-5 [31]. Results of the present study revealed that LNCaP cells express the conventional TbR-I mRNA at a level lower than approximately one copy per two cells, suggesting that ALK-5 is not the functional TbR-I in these cells. More recently, LNCaP cells have been shown to have a defect in the ALK-5 gene [23]. Thus, the undetectable level of ALK-5 is likely due to a fundamental change at the gene level. Despite the apparent absence of ALK-5 expression, LNCaP cells do respond to TGFb1 under certain culture conditions. Specifically, results of the present study, coupled with those of previous reports [20], have indicated that these cells are sensitive to TGF-b1 when a proliferative dose of DHT (0.1 nM) is added to the culture medium. In addition, TGF-b1 inhibited the proliferative effects of EGF and TGF-a in LNCaP cells [21]. These observations suggest an alternative possibility regarding the mechanism of action of TGF-b receptors in that a heteromeric complex of TbR-II and TbR-I may not be necessary for TGF-b1 signaling and that TbR-II alone may be able to transduce signal for TGF-b1 in LNCaP cells. However, such possibility seems unlikely because the presence of the heteromeric complex composed of TbR-I and TbR-II has been demonstrated in multiple systems [32–34]. Moreover, a heteromeric complex composed of type I and type II receptor is required for signaling for other members of TGF-b superfamily [28–30]. Since it is likely that a heteromeric complex composed of TbR-I and TbR-II is necessary for TGF-b signaling, results of the present study suggest that there may be more than one form of functional TbRI. In the present study, attempts to demonstrate a heteromeric complex of type I and type II receptors by 125 I-TGF-b1 cross-linking assay were not successful (data not shown). It may be that LNCaP cells contain TGF-b receptors that are relatively refractory to cross-linking with ligand, as is the case with some activin receptors [9]. Nevertheless, LNCaP cells were demonstrated to respond to TGF-b1 in the apparent

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FIG. 5. Effect of DHT on the expression of ALK-1 and -2 in LNCaP cells. (A) Western blot analysis. Cells were cultured in varying concentrations of DHT for 4 days. Media were changed once on day 2. Subsequently, cells were harvested and lysed using protein sample buffer. Then, 100 mg of each protein sample was separated by electrophoresis in 10% SDS–polyacrylamide gel and immunoreactive bands were visualized by enhanced chemiluminescence. (B) Competitive quantitative RT-PCR. For each analysis, 200 ng of poly(A) RNA was used. The competitor was generated by the previously described low-stringency method. For each reaction, 80 fg of each competitor was used. Products were separated by electrophoresis using 1% agarose gel and visualized by staining with ethidium bromide. PC3 cells were used as a positive control. To control for genomic DNA contamination, all reactions were carried out in the absence of reverse transcriptase. DHT did not significantly change the levels of expression of ALK-1 and -2 both at the protein and at the mRNA level. (RT) Reverse transcriptase.

absence of the conventional TbR-I, ALK-5. Since TGFb signaling requires both TbR-I and TbR-II, these results suggest that there may be a heterogeneous population of TbR-I. As an initial attempt to identify the functional TbRI in LNCaP cells, the expression of three proteins, ALK1, -2, and -4, was investigated. Published reports have demonstrated that these proteins bind TGF-b1 in vitro [11–14]. Of these putative receptors, ALK-1 and -2 were detected in LNCaP cells by RT-PCR and Western blot analysis. When the effect of DHT on the levels of expression of ALK-1 and -2 was investigated by Western blot analysis and competitive quantitative RTPCR, the results demonstrated that the TGF-b1-sensitive dose of DHT did not change the levels of expression of these receptors in LNCaP cells; there was a relatively high but constant level of these putative receptors in these cells under all concentrations of DHT investigated in this study. Thus, it is likely that the level

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of TbR-I in LNCaP cells is maintained constitutively at a level that is sufficient to transduce signal for TGFb, rendering the level of TbR-II as the rate-limiting factor concerning TGF-b sensitivity in LNCaP cells. Further studies are under way to verify this concept. The possibility remains that the functional TbR-I in LNCaP cells may be protein(s) other than ALK-1 or -2. Recently, Yamashita et al. have demonstrated that a rat pituitary tumor cell line, GH3 , has a heterogeneous TbR-I band (Ç69–74 kDa) following cross-linking with 125 I-TGF-b1 [35]. Further analysis showed that these cells express unidentified components capable of forming a complex of 72–74 kDa with 125I-TGF-b1; the protein(s) was different from ALK-1, -2, -4, and -5. At present, it remains to be elucidated whether this unknown protein is a functional TGF-b receptor. In conclusion, results of the present study demonstrated that TGF-b1 signaling in LNCaP cells can be manifested in the apparent absence of the conventional

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TbR-I, ALK-5. Since TGF-b signaling requires a heteromeric complex composed of TbR-I and TbR-II, it is likely that there are additional functional TbR-Is. In LNCaP cells, it is likely that the functional TbR-I is either ALK-1 or -2, or both. Nevertheless, the possibility remains that the functional TbR-I in these cells may be protein(s) other than ALK-1 and -2. Future studies will focus on determining the role of ALK-1 and -2 in mediating TGF-b1 signaling in these cells. We thank Drs. Kohei Miyazono (The Cancer Institute, Tokyo, Japan), Anita Roberts (NCI, Bethesda, MD), Seong Jin Kim (NCI, Bethesda, MD), Peter ten Dijke (Ludwig Institute for Cancer Research, Uppsala, Sweden), and Rik Derynck (UCSF, San Francisco, CA) for helpful discussions. We also thank Dr. Joan Massague (Memorial Sloan-Kettering Cancer Center, New York, NY) for kindly providing p3TP-Lux. Antibodies were kindly provided by Dr. Miyazono.

REFERENCES 1. Kingsley, D. M. (1994). The TGF-b superfamily: New members, new receptors, and new genetic tests of function in different organisms. Genes Dev. 8, 133–146. 2. Massague, J., Cheifetz, S., Laiho, M., Ralph, D. A., Weis, F. M. B., and Zentella, A. (1992). Transforming growth factorb. Cancer Surv. 12, 81–103. 3. Wilding, G. (1991). Response of prostate cancer cells to peptide growth factor: Transforming growth factor-b. Cancer Surv. 11, 147–163. 4. Derynck, R. (1994). TGF-b-mediated signaling. Trends Biochem. Sci. 19, 548–553. 5. Lopez-Casillas, F., Cheifetz, S., Doody, J., Andres, J. L., Lane, W. S., and Massague, J. (1991). Structure and expression of the membrane proteoglycan betaglycan, a component of the TGFb receptor. Cell 67, 785–795. 6. Lin, H. Y., Wang, X., Ng-Eaton, E., Weinberg, R. A., and Lodish, H. F. (1992). Expression cloning of the TGF-b type II receptor, a functional transmembrane serine/threonine kinase. Cell 68, 775–785. 7. Franzen, P., ten Dijke, P., Ichijo, H., Yamashita, H., Schulz, P., Heldin, C., and Miyazono, K. (1993). Cloning of a TGF-b type I receptor that forms a heteromeric complex with the TGF-b type II receptor. Cell 75, 681–692. 8. Bassing, C. H., Yingling, J. M., Howe, D. J., Wang, T., He, W. W., Gustafson, M. L., Shah, P., Donahoe, P. K., and Wang, X.-F. (1994). A transforming growth factor b type I receptor that signals to activate gene expression. Science 263, 87–89. 9. Wrana, J. L., Attisano, L., Carcamo, J., Zentella, A., Doody, J., Laiho, M., Wang, X., and Massague, J. (1992). TGF-b signals through a heteromeric protein kinase receptor complex. Cell 71, 1003–1014. 10. Wrana, J. L., Attisano, L., Wieser, R., Ventura, F., and Massague, J. (1994). Mechanism of activation of the TGF-b receptor. Nature 370, 341–347. 11. ten Dijke, P., Yamashita, H., Ichijo, H., Franzen, P., Laiho, M., Miyazono, K., and Heldin, C.-H. (1994). A transforming growth factor b type I receptor that signals to activate gene expression. Science 264, 101–104. 12. ten Dijke, P., Ichijo, H., Franzen, P., Schulz, P., Saras, J., Toyoshoma, H., Heldin, C.-H., and Miyazono, K. (1993). Activin re-

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04-24-98 13:44:06

159

ceptor-like kinases: a novel subclass of cell-surface receptors with predicted serine/threonine kinase activity. Oncogene 8, 2879–2887. 13. Ebner, R., Chen, R.-H., Shum, L., Lawler, S., Zioncheck, T. F., Lee, A., Lopez, A. R., and Derynck, R. (1993). Cloning of a type I TGF-b receptor and its effect on TGF-b binding to the type II receptor. Science 260, 1344–1348. 14. Attisano, L., Carcamo, J., Ventura, F., Weis, F. M. B., Massague, J., and Wrana, J. (1993). Identification of human activin and TGF-b type I receptors that form heteromeric kinase complexes with type II receptors. Cell 75, 671–681. 15. Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr, J. P., Rosenthal, H., Chu, M. T., Mirand, E. A., and Murphy, G. P. (1981). LNCaP model of human prostatic carcinoma. Cancer Res. 43, 1808–1817. 16. Wilding, G., Chen, M., and Gelman, E. (1989). Aberrant response in vitro of hormone-responsive prostate cancer cells to anti-androgens. Prostate 14, 103–115. 17. Kokontis, J., Kiyoshi, I., Hipakka, R. A., and Liao, S. (1991). Expression and function of normal and LNCaP androgen receptors in androgen-insensitive human prostatic cancer cells. Receptor 1, 271–279. 18. Sonnenschein, C., Olea, N., Pasanen, M. E., and Soto, A. M. (1989). Negative controls of cell proliferation: human prostate cancers and androgens. Cancer Res. 49, 3474–3481. 19. Lee, C., Sutkowski, D. M., Sensibar, J. A., Zelner, D., Kim, I., Amsel, I., Shaw, N., Prins, G. S., and Kozlowski, J. M. (1995). Regulation of proliferation and production of prostate specific antigen in androgen sensitive prostatic cancer cells, LNCaP, by dihydrotestosterone. Endocrinology 136, 796–803. 20. Kim, I. Y., Kim, J.-H., Zelner, D. J., Ahn, H.-J., Sensibar, J. A., and Lee, C. (1996). Transforming growth factor-b1 is a mediator of androgen-regulated growth arrest in an androgen-responsive prostatic cancer cell line, LNCaP. Endocrinology 137, 991–999. 21. Kim, I. Y., Zelner, D. J., Sensibar, J. A., Ahn, H.-J., Park, L., Kim, J.-H., and Lee, C. (1995). Modulation of sensitivity to transforming growth factor-b1 (TGF-b1) and the level of type II TGF-b receptor in LNCaP cells by dihydrotestosterone. Exp. Cell Res. 222, 103–110. 22. Schuurmans, A. L. G., Bolt, J., Veldscholte, J., and Mulder, E. (1991). Regulation of growth of LNCaP human prostate tumor cells by growth factors and steroid hormones. J. Steroid Biochem. Mol. Biol. 40, 193–197. 23. Kim, I. Y., Ahn, H.-J., Zelner, D. J., Shaw, J. W., Sensibar, J. A., Kim, J.-H., Kato, M., and Lee, C. (1996). Genetic change in transforming growth factor-b (TGF-b) receptor type I gene correlates with insensitivity to TGF-b1 in human prostate cancer cells. Cancer Res. 56, 44–48. 24. Siebert, P. D., and Larrick, J. W. (1992). Competitive PCR. Nature 359, 557–558. 25. Forster, E. (1994). Rapid generation of internal standards for competitive PCR by low-stringency primer annealing. Biotechniques 16, 1006–1008. 26. Steele, R. E. D., and Torrie, J. H. (1960) ‘‘Principles and Procedures of Statistics,’’ McGraw-Hill, New York, NY. 27. Bouaboula, M., Legoux, P., Pessegue, B., Delpech, B., Dumont, X., Piechaczyk, M., Lasellas, P., and Shire, D. (1992). Standardization of mRNA titration using a polymerase chain reaction method involving co-amplification with multispecific internal control. J. Biol. Chem. 267, 21830–21838. 28. Ruberte, E., Marty, T., Nellen, D., Affolter, M., and Basler, K. (1995). An absolute requirement for both the type II and type

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KIM, ZELNER, AND LEE I receptors, punt and thick veins, for dpp signaling in vivo. Cell 80, 889–897.

29. Liu, F., Ventura, F., Doody, J., and Massague, J. (1995). Human type II receptor for bone morphogenic proteins (BMPs): Extension of the two kinase receptor model to the BMPs. Mol. Cell. Biol. 15, 3479–3486. 30. Wrana, J. L., Tran, H., Attisano, L., Arora, L., Childs, S. R., Massague, J., and O’Connor, M. B. (1994). Two distinct transmembrane serine/threonine kinases from drosophila melanogaster form an activin receptor complex. Mol. Cell. Biol. 14, 944–950. 31. Miettinen, P. J., Ebner, R., Lopez, A. R., and Derynck, R. (1994). TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: Involvement of type I receptors. J. Cell Biol. 127, 2021–2036.

32. Ito, M., Yasui, W., Kyo, E., Yokozaki, H., Nakayama, H., Ito, H., and Tahara, E. (1992). Growth inhibition of transforming growth factor b on human gastric carcinoma cells: Receptor and post-receptor signaling. Cancer Res. 52, 295–300. 33. Yamashita, H., ten Dijke, P., Franzen, P., Miyazono, K., and Heldin, C.-H. (1994). Formation of hetero-oligomeric complexes of type I and type II receptor for transforming growth factor-b. J. Biol. Chem. 269, 20172–2018. 34. Segarini, P. R., Rosen, D. M., and Seyedin, S. M. (1989). Binding of transforming growth factor-beta to cell surface proteins varies with cell type. Mol. Endocrinol. 3, 261–272. 35. Yamashita, H., Okadome, T., Franzen, P., ten Dijke, P., Heldin, C.-H., and Miyazono, K. (1995). A rat pituitary tumor cell line (GH3) expresses type I and type II receptors and other cell surface binding protein(s) for transforming growth factor-b. J. Biol. Chem. 270, 770–774.

Received October 7, 1997 Revised version received January 28, 1998

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