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Originally published In Press as doi:10.1074/jbc.M511781200 on January 20, 2006

J. Biol. Chem., Vol. 281, Issue 12, 7765-7774, March 24, 2006
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Novel Permissive Role of Epidermal Growth Factor in Transforming Growth Factor beta (TGF-beta) Signaling and Growth Suppression

MEDIATION BY STABILIZATION OF TGF-beta RECEPTOR TYPE II*Formula

Kyung Song, Tracy L. Krebs, and David Danielpour1

From the The Case Comprehensive Cancer Center, The Division of General Medical Sciences and the Department of Pharmacology, Case School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106

Received for publication, November 1, 2005 , and in revised form, January 13, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor beta (TGF-beta) signals through TGF-beta receptor serine/threonine kinases (TbetaRI and TbetaRII) and Smads, regulating cell growth and apoptosis. Although loss of TGF-beta receptor levels is strongly selected for during the progression of most cancers, tumor cells frequently escape from complete loss of TGF-beta receptors through unknown mechanisms. Here, we provide the first evidence that epidermal growth factor (EGF) signaling, which is generally enhanced in cancer, is permissive for regulation of gene expression and growth suppression by TGF-beta in LNCaP prostate adenocarcinoma cells. Our results support that these permissive effects occur through enhanced stability of TbetaRII mRNA and reversal of TGF-beta-mediated TbetaRII mRNA loss. Changes in stability of TbetaRII mRNA occur soon after EGF or TGF-beta1 addition (optimal within 3 h) and are independent of de novo protein synthesis or transcription. Remarkably, such loss of TbetaRII by TGF-beta can be mediated by a kinase-dead TbetaRII (K277R), as well as by other forms of this receptor harboring mutations at prominent autophosphorylation sites. Moreover, Smad3 small interfering RNA, which blocks TGF-beta-induced AP-1 promoter activity, does not block changes in the expression of TbetaRII by EGF or TGF-beta. We have also shown that changes in TbetaRII levels by EGF are EGF receptor-kinase-dependent and are controlled by signals downstream of MEK1/2. Our findings provide invaluable insights on the role of the EGF receptor-kinase in enhancing TGF-beta responses during prostate carcinogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor-betas (TGF-beta)2 are multifunctional cytokines that regulate cell cycle arrest, mitosis, differentiation, and apoptosis (1–9). As one of the earliest events in the cellular responses to these multifunctional growth factors, TbetaRI and TbetaRII cell surface signaling receptors form a heterotetrameric complex upon binding TGF-betas (10–12). The constituitively active kinase domain of TbetaRII activates TbetaRI by transphosphorylation of the GS box in the cytoplasmic domain (13). The activated TbetaRI then propagates TGF-beta signals partly by activating Smads 2 and 3 by phosphorylating their C-terminal serines (14–16). These Smads then translocate to the nucleus where they function either directly as transcription factors or indirectly as transcription co-modulators (16–18).

TGF-beta receptors, which function as tumor suppressors in normal and preneoplastic tissues, acquire oncogenic functions during tumor progression. TGF-beta receptors are mutated or/and expressed at substantially reduced levels in a variety of human cancers including colon, gastric, and prostate cancers, correlating with acquisition of resistance to growth suppression by TGF-beta (19–22). Significantly, restoration of TGF-beta receptor expression or function can reverse the malignant phenotype in a variety of carcinoma cells (3, 23). Consistent with those observations, suppression of TGF-beta signaling by dominant-negative TbetaRII has been shown to promote malignant transformation of non-tumorigenic cell lines (4, 6).

Despite the apparent selective pressure for carcinomas to lose TGF-beta receptors, there appears to be resistance against complete loss of those receptors during the progression of many carcinomas (24), consistent with the conversion of the function of TGF-beta to that of an oncogene (25). Thus, the molecular mechanism behind the switch of the function of TGF-beta from tumor suppressor to tumor promoter is likely to require a signal for the retention of TGF-beta receptors during carcinogenesis.

EGF is a 6-kDa polypeptide that binds to a 170-kDa transmembrane tyrosine kinase receptor (EGFR) expressed on a wide variety of normal and neoplastic cells. Binding of EGF to EGFR causes receptor dimerization and autophosphorylation, leading to activation of a number of downstream signal transduction pathways, such as Ras/Raf/mitogen-activated protein kinase and PI3-kinase/Akt, that mediate cell proliferation, angiogenesis, and apoptosis by EGF (26). Although EGFR appears to be amplified and activated in many cancers, its role in the malignant phenotype is not entirely clear (27). In most cells EGF is growth stimulatory and anti-apoptotic; however, a number of tumor cell lines have been shown to be killed by this peptide through unknown mechanisms (28, 29). Interestingly, EGFR and its homologue HER2 have been reported to control various TGF-beta responses both in vitro and in vivo (30–34).

In this study we report the first evidence that the EGF signaling pathway may enhance TGF-beta responses through increasing the stability of TbetaRII, as demonstrated with the most widely used human prostate adenocarcinoma cell model, LNCaP. Although seemingly counterintuitive, the activation of TGF-beta responses by EGF is consistent with the general induction of TGF-beta ligand by this mitogen (35–37) as well as a requirement of AP-1 and MEK1/2 for many TGF-beta responses, including growth suppression (38–41). Elucidation on how EGF can stabilize TbetaRII mRNA is thus likely to provide mechanistic insight on the conversion of the function of TGF-beta from tumor suppressor to tumor promoter.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Sources were: LNCaP, DU-145, PC-3 (ATTC); recombinant human TGF-beta1 and TGF-beta2 (R&D Systems); anti-phospho Smad3 antibody (catalog number 9514), anti-phospho-p44/42 mitogen-activated protein kinase (Thr-202/Tyr-204) ((catalog number 9106), anti-phospho-p70S6K (Thr-389) (catalog number 9205), anti-phospho-c-Jun (Ser-63) (catalog number 9261) (Cell Signaling); anti-TbetaRII (sc-1700) and anti-Smad3 (sc-8332) (Santa Cruz Biotechnology); peptide-N-glycosidase F (New England Biolabs); DMEM/F12 (1:1, v/v) (Invitrogen); mouse EGF (BioSource International); PD153035, AG1478, SB202190, U0126, PD98059, LY294002, and SP600125 (Calbiochem); rapamycin (BioMol); characterized FBS and DC-stripped FBS (HyClone); pUSE-act-Ras (Upstate Cell Signaling Solutions); and pFC-MEK1, pFC-Elk1, AP-1-luciferase, pFC-MEK1, cis-acting PathDetectTM constructs (Stratagene). For the pCMV5-c-Fos expression vector, the full-length coding region of c-Fos was amplified using HighFidelityTM (Roche Applied Science) PCR from the pLIB-prostate human cDNA library (Clontech) and inserted into the pCMV5 vector.

Cell Culture—LNCaP cells were maintained in DMEM/F12 containing 10% FBS in poly-D-lysine-coated 75-cm2 culture flasks (42). In all experiments involving LNCaP cells, unless indicated, cells were attached to polylysine-coated dishes and cultured overnight in DMEM/F12 supplemented with 1% DC-stripped FBS and 15 mM HEPES (42).

In general, all signaling inhibitors were added to cells 1–2 h before the addition of EGF (20 ng/ml), and cells were incubated in the absence or presence of TGF-beta1 (10 ng/ml) for up to 48 h. In all time course experiments, the times of EGF or TGF-beta addition were varied, with total culture times and all other conditions kept constant for proper control. Vehicles used as controls for TGF-beta1 and EGF (4 mM HCl, 1 mg/ml bovine serum albumin, diluted 1000-fold in assay) were shown to have no effect on TbetaRII level at all treatment times.

TGF-beta Sandwich Enzyme-linked Immunosorbent Assays—TGF-beta proteins were measured by established sandwich enzyme-linked immunosorbent assays (43, 44). LNCaP cells were cultured under serum-free conditions (42) and then treated with various combinations of growth factors and hormones. Serum-free conditions were used for the preparation of conditioned medium to avoid carryover of latent TGF-betas in serum (43). Following either 24 or 48 h of conditioning, medium was assayed for TGF-beta. Daily production rates of TGF-beta were normalized to the DNA content of producer cells (42).

siRNA—Human Smad3 siRNA oligonucleotide (si-Smad3, 5'-GGCCATCACCACGCAGAACdTdT-3') and its complementary RNA strand were synthesized by Dharmacon, annealed in vitro, and co-transfected into LNCaP cells (80 nM/well in 6-well plates) with indicated plasmids by Lipofectamine-PlusTM (Invitrogen) according to the manufacturer's protocol. A 19-mer scrambled siRNA pair without a known human mRNA target was used as a negative control. Cell extracts were prepared 48 h after siRNA transfection.

Adenovirus Gene Delivery—An adenovirus vector that directs the expression of WT-TbetaRII (AdMax-WT-TbetaRII) was constructed using the AdMax system (Microbix Biosystems). The full-length coding sequence of human TbetaRII (1.7 kb) excised from pCMV5-HA-TbetaRII (45) was subcloned into the pDC515 adenovirus shuttle vector. Human embryonic kidney 293 cells were co-transfected with 1 µg of pDC515-HA-TbetaRII and 1 µg of genomic vector pBHGfrtDE1,3FLP in 6-well plates, using a standard calcium phosphate precipitation method (42). Following transfection, the cells were maintained in DMEM/F12 containing 2% FBS for ~10 days or until the appearance of viral cytopathic effects. Cells were then lysed by four serial freeze-thaw cycles (on dry ice and 37 °C). Liberated viral particles were further amplified by two-three serial infections through human embryonic kidney 293 cells, according to the manufacturer's protocol. To titer total viral particles, aliquots of virus stocks were diluted 20-fold in lysis solution (0.1% sodium dodecylsulfate, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA) and incubated for 10 min at 56 °C. The optical density of the samples at 260 nm was used to calculate virus content using the relationship 1.1 x 1012 virus particles/ml/A260 units. All viral preparations were evaluated for expression of recombinant protein by Western blot analysis of transduced NRP-154 and LNCaP cells.

For experiments involving use of adenovirus, LNCaP cells were infected overnight with 1.0–2.5 x 1010 viral particles/2 ml/well (in 6-well dishes). Their medium was then replaced to remove residual virus particles before treatment with the indicated factors.

Northern Blot Analysis—Northern blot analysis was performed essentially as described (46, 47). In brief, 10 µg of total RNA was electrophoresed and equal loading and even transfer were assessed by visualization of the 18 and 28 S rRNAs. The presence of indicated mRNA was detected with cDNA probes labeled with [32P]dCTP using PrimeIt® RmT random primer labeling kit (Stratagene).

RT-PCR—RT was performed as described (6). The PCR primers applied to detect TbetaRII expression were 5'-AGCACGATCCCACCGCACGTTCAGAAG-3' (forward) and 5'-CTATTTGGTAGTGTTTAGGGAGCCGT-3' (reverse) and yielded a 1.7-kb fragment. Taq polymerase master mix (Promega) was used for PCR amplification of TbetaRII from 0.1 µg of LNCaP cDNA template, using 32 cycles of the following temperature gradients: 95 °C for 15 s, 63 °C for 30 s, and 72 °C for 2 min. beta-Actin, amplified as above for 21 cycles, served as an internal control.

Western Blot Analysis—Western blot analysis was performed essentially as described (6, 46). Cells were lysed in cold radioimmune precipitation assay buffer containing a protease inhibitor mixture (Roche Applied Science) supplemented with 1 mM phenylmethylsulfonyl fluoride, 2.5 mM sodium pyrophosphate, and 1 mM beta-glycerophosphate. The resulting lysates were clarified in 1.5-ml tubes by centrifugation at 16,000 x g for 10 min, and supernatants were quantified by a microtiter BCA protein assay (Pierce) as described before (46). For determination of TbetaRII levels, aliquots of 10–20 µg (protein) were deglycosylated by incubation with peptide-N-glycosidase F according to the manufacturer's instructions (New England Biolabs).

Transient Transfection and Luciferase Assay—All of these procedures were performed essentially as described (42, 46). In brief, LNCaP cells were plated overnight at a density of 1.25 x 105 cells/1 ml/well in 12-well plates. Reporter constructs, 1–2µg, were co-transfected with 12.5–25 ng of CMV-Renilla reporter construct using Lipofectamine-PlusTM followed by treatment with EGF (20 ng/ml) and TGF-beta1 (10 ng/ml). Luciferase activity was measured using a dual luciferase assay kit (Promega) and a ML3000 microtiter plate luminometer. All luciferase activity was expressed as normalized values of firefly luciferase to Renilla luciferase.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
EGF Permits TGF-beta1 Autoinduction and Growth Suppression in LNCaP Cells—Major interest in the role of TGF-beta in the prostate is attributed to the ability of this cytokine to induce cell death, suppress tumor growth, and likely mediate androgen ablation-induced cell death (49, 50). We used the LNCaP cell line, which expresses low to undetectable levels of TGF-beta ligands and receptors, to identify factors that may suppress tumor growth by their ability to up-regulate TGF-beta ligands or receptors. We first screened a variety of growth factors and hormones for their ability to enhance expression of TGF-beta ligands in these cells under serum-free conditions. Of these, only EGF was able to enhance the expression of TGF-beta1 to a level detectable by the most sensitive and specific TGF-beta sandwich enzyme-linked immunosorbent assays reported (43). Following a secondary screen of factors that could synergize with EGF to enhance TGF-beta1 expression, we found that only TGF-beta ligands (TGF-betas 1, 2, or 3) elevated TGF-beta1 expression in the presence of EGF (42). We now show that EGF is clearly permissive for the autoinduction of TGF-beta1 expression as measured by enzyme-linked immunosorbent assay (Fig. 1A) and Northern blot analysis (Fig. 1, B and C). Use of TGF-beta2 treatment rather than TGF-beta1 confirmed that changes in TGF-beta1 protein levels measured in conditioned medium reflected only that made by LNCaP cells.


Figure 1
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  		FIGURE 1.
EGF is permissive to TGF-beta-induced TGF-beta1, c-Fos, and Egr-1 expression in LNCaP cells. A, effect of EGF (20 ng/ml), TGF-beta2 (2.5 ng/ml), or combination of EGF and TGF-beta2 on TGF-beta1 production rate (48) in serum-free medium (48 h). Data shown are averages (± S.D.) of triplicate independent measurements. B and C, EGF permits autoinduction of TGF-beta1 at mRNA levels. D, EGF permits TGF-beta1-enhanced expression of c-Fos and Egr-1 mRNAs. Total RNA (10 µg) was subjected to Northern blot analysis. Representative blots of three independent experiments are shown (B–D).

 
The AP-1 complex (commonly composed of dimers of c-Jun and c-Fos, or JunD/Fra-2) and Egr-1 have been shown to mediate TGF-beta1 autoinduction by activating its transcription (51, 52). Thus, to understand how EGF permits autoinduction of TGF-beta1 in LNCaP cells, we studied the individual and combined effects of TGF-beta1 and EGF on c-Fos, c-Jun, Egr-1, and EGFR mRNA expression. Co-treatment of these cells with TGF-beta1 and EGF induced c-Fos and Egr-1 mRNA levels, which peaked by 48 h. At this time point, EGF alone induced expression of both these mRNAs. In contrast, TGF-beta1 alone (without EGF) was ineffective in modulating these transcription factors. However, TGF-beta1 was able to induce the expression of both these mRNAs when this cell line was treated together with EGF (Fig. 1D). Neither TGF-beta1 nor EGF added alone or together enhanced the mRNA levels of c-Jun or EGFR. These results show that EGF is permissive not only for TGF-beta1 autoinduction but also for TGF-beta's induction of c-Fos and Egr-1.


Figure 2
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  		FIGURE 2.
EGF enhances the ability of TGF-beta1 to inhibit growth or induce death of LNCaP cells. LNCaP cells (5 x 104 cells/well/1.0 ml of DMEM/F12 + 10% DC-FBS) were plated in 12-well dishes, treated with EGF (20 ng/ml) or vehicle, and 24 h later treated with TGF-beta1 (10 ng/ml) or vehicle. Cell numbers were assayed 3, 6, 9, and 12 days later using a Coulter Electronics counter. Data shown are averages (±S.D.) of triplicate independent measurements.

 


Figure 3
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  		FIGURE 3.
EGF is permissive to the transactivation of the PAI-1 promoter construct, 3TP-luciferase, by TGF-beta1 through a mechanism that involves TbetaRII expression and the elevation of endogenous TbetaRII mRNA. A, LNCaP cells were plated in 1% DC-FBS + DMEM/F12 and transiently co-transfected with 3TP-Lux and CMV-Renilla. B, LNCaP cells were transiently transfected with 3TP-lux, CMV-Renilla plasmids, and either pMFG-neo empty vector or pMFG-neo-DN-TbetaRII (dominant-negative TbetaRII). Cells were then treated with EGF (20 ng/ml), TGF-beta1 (10 ng/ml), or both EGF and TGF-beta1, and luciferase activity was measured 48 h later. Data represent averages of triplicate independent measurements (±S.E.) of firefly luciferase/Renilla luciferase readings normalized to untreated controls (A, B). C, endogenous TbetaRII mRNA in cells not expressing exogenous TbetaRII, but treated with EGF or vehicle for 24 h, was determined by RT-PCR using primers that yield a 1.7-kb fragment. All data are representative of three independent experiments.

 


Figure 4
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  		FIGURE 4.
EGF does not enhance TbetaRII promoter activity. A–C, LNCaP cells were plated in 1% DC-FBS + DMEM/F12 and transiently co-transfected with CMV-Renilla and the full-length (–1690/+38) (A) or the deletion (–216/+35) (B, C) of TbetaRII promoter-luciferase. 20 ng/ml of EGF was added after transfection, and 10 ng/ml of TGF-beta1 was added 16 h after. Cells were then incubated for an additional 48 h before dual luciferase assay (A, B). Separately, cells were incubated in the absence or presence of 1 µM MS-275 before being assayed (C). A dual luciferase assay was performed; data shown are averages (±S.E.) of triplicate independent measurement of firefly luciferase/Renilla luciferase readings normalized to untreated controls (A–C). All data are representative of three independent experiments.

 
We next performed a cell growth assay to examine whether the permissive effect of EGF also occurred at the level of growth inhibition by TGF-beta1. LNCaP cells, plated in DMEM/F12 containing 10% DC-FBS to sustain cell proliferation, were pretreated with or without EGF for 24 h prior to TGF-beta1 addition. Cell numbers were determined at days 3, 6, 9, and 12 following the treatment of TGF-beta1. Cells cultured with TGF-beta1 alone were growth arrested relative to controls that showed a slight increase in proliferation. However, when pretreated with EGF, TGF-beta1 promoted death of LNCaP cells, whereas EGF alone actually stimulated growth (Fig. 2). Thus, in LNCaP cells EGF enhances the ability of TGF-beta to suppress growth arrest and appears to be permissive to TGF-beta-induced cell death/apoptosis.

Endogenous TbetaRII mRNA Is Enhanced by EGF through a Non-transcriptional Mechanism—To define the mechanism by which EGF can permit TGF-beta1 responses we next studied the effects of EGF on TGF-beta-regulated transcriptional activity, using a highly TGF-beta-responsive plasminogen activator inhibitor-1 promoter-luciferase reporter construct, 3TP-lux. We showed that neither TGF-beta1 nor EGF alone activated 3TP-lux, whereas co-treatment with both of these agents induced this promoter activity ~7-fold (Fig. 3A). Consistent with a TGF-beta receptor-mediated response, this induction was lost by co-expression of dominant-negative TbetaRII (Fig. 3B). However, LNCaP cells are reported to be weakly responsive to TGF-beta1 due to very low expression of TbetaRII (53, 54). Not surprisingly, we were unable to detect TbetaRII expression in these cells even after EGF treatment at the mRNA level by Northern blot or at the protein level by a significantly enhanced Western blot procedure (6). However, the increased sensitivity of detection offered by semi-quantitative RT-PCR revealed that TbetaRII was elevated >4-fold following EGF treatment (Fig. 3C), indicating that EGF may enhance TGF-beta responses through up-regulating TbetaRII levels.


Figure 5
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  		FIGURE 5.
Retention of the permissive activity of EGF on TGF-beta responses in LNCaP cells overexpressing TbetaRII. A–D, LNCaP cells were plated in 1% DC-FBS + DMEM/F12 and transiently co-transfected with CMV-Renilla along with 3TP-Lux (A), p21 promoter-luciferase (B), or AP-1-Luc (C, D) in the presence of pCMV5-TbetaRII or pCMV5 (0.5 or 1.0 µg). E, transcriptional activity was determined followed by co-transfection with 3TP-Lux, CMV-Renilla, and 1 µg of either pCMV5 empty vector or pCMV5-TbetaRI (ALK5). 20 ng/ml of EGF was added after transfection, and 10 ng/ml of TGF-beta1 was added 16 h after. Cells were then incubated either for an additional 48 h or the indicated times before being assayed (A–E). Data shown are averages (±S.E.) of triplicate independent measurements of firefly luciferase/Renilla luciferase readings normalized to untreated controls (A–E). All data are representative of three independent experiments.

 
Enhanced expression of TbetaRII mRNA could occur either through a transcriptional mechanism or through stabilization of the TbetaRII message. The former possibility was tested by measuring TbetaRII promoter activity. LNCaP cells were transiently transfected with the full-length (–1690/+38) or truncated (–216/+35) TbetaRII promoter-luciferase constructs, followed by treatment with EGF, TGF-beta1, or both EGF and TGF-beta1 (Fig. 4, A and B). Although TGF-beta1 enhanced (<2-fold) the activity of only the full-length TbetaRII promoter construct, EGF had no effect on either construct whether in the presence or absence of TGF-beta1 (Fig. 4, A and B). MS-275, a histone deacetylase inhibitor used as a positive control (55), enhanced activity of TbetaRII (–216/+35) promoter construct by 8-fold (Fig. 4C). These results suggest that induction of endogenous TbetaRII mRNA expression by EGF (shown in Fig. 3C) occurs through a non-transcriptional mechanism involving enhanced stability of TbetaRII mRNA.


Figure 6
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  		FIGURE 6.
EGF rapidly enhances the levels of endogenous and of exogenously expressed TbetaRII protein and mRNA. LNCaP cells plated with 1% DC-FBS + DMEM/F12 were infected with a recombinant adenovirus expressing wild-type TbetaRII (AdMax-WT-TbetaRII) (1:200) for 24 h. Medium was replaced (without virus) before various times of treatment with 20 ng/ml of EGF (A and B), and cells were harvested for assaying TbetaRII by either Western blot (A) or by Northern blot (B) analyses. C, effect of EGF on the endogenous level of TbetaRII in LNCaP was measured by RT-PCR in a time course experiment similar to that in panel B but without adenoviral infection. In the above time course experiments, the time of EGF addition was varied, with total culture times and all other conditions kept constant for proper control. All data are representative of three independent experiments. Deglycosylated protein (20 µg) and total RNA (10 µg) were subjected to Western blot and Northern blot analyses, respectively (A, B). A representative blot of three independent experiments is shown (A–C).

 
EGF Permits TGF-beta1-induced Transcriptional Responses in Cells Overexpressing Exogenous TbetaRII—Consistent with our hypothesis that enhanced stabilization of TbetaRII mRNA mediates the permissive effect of EGF on TGF-beta responses, EGF can also enhance TGF-beta responses in LNCaP cells that overexpress TbetaRII via transient transfection of a CMV-driven promoter construct, pCMV5-TbetaRII (Fig. 5A). Although transfection of this TbetaRII expression vector enabled TGF-beta-induced 3TP-lux activity without EGF, cells overexpressing TbetaRII retained their exquisite sensitivity to EGF for potentiating this TGF-beta response (Fig. 5A). Moreover, EGF also permitted TGF-beta1-induced p21CIP1/WAF-1 transcriptional activity 9-fold (Fig. 5B) and enhanced TGF-beta1-induced AP-1 basic response element promoter-luciferase construct (AP-1-luc) >40-fold in LNCaP cells overexpressing TbetaRII, showing maximum activity at 48 h (Fig. 5, C and D). In contrast to TbetaRII, overexpression of TbetaRI (ALK5) did not enhance TGF-beta responses either with or without EGF (Fig. 5E). Taken together, these results indicate that LNCaP cells retain the permissive effect of EGF even after overexpression of TbetaRII by a CMV-driven promoter and further support that this EGF effect is mediated by changes in TbetaRII mRNA stability.

EGF Stabilizes TbetaRII Expression—We tested our hypothesis that EGF enhances TGF-beta responses through elevation of TbetaRII levels, using LNCaP cells ectopically overexpressing TbetaRII delivered by either transient transfection or adenoviral infection. Transfected cells were treated with EGF, TGF-beta1, EGF+TGF-beta1, or vehicle, and 24–48 h later levels of TbetaRII were measured by Western blot analysis. As expected, TbetaRII was significantly elevated by EGF in cells transfected with pCMV5-TbetaRII or infected with AdMax-TbetaRII (Fig. 6). In time course experiments with these cells, protein levels of this receptor were enhanced by 3 h of EGF treatment, reached a peak at 6 h, and persisted at that level for over 24 h (Fig. 6A). To test whether the changes in TbetaRII protein levels reflected changes in the expression of TbetaRII mRNA, total RNA from LNCaP cells treated similarly was subjected to Northern blot analysis. In this experiment expression of TbetaRII mRNA was increased as early as 1 h of EGF treatment, and robust (>20-fold) increases in this message appeared between 3 and 6 h after EGF treatment. Thereafter, levels of TbetaRII mRNA dropped for the next 3–6 h and then kept steady up to 24 h (Fig. 6B). The levels of endogenous TbetaRII mRNA (detected by RT-PCR) in LNCaP cells that were not infected or transfected with TbetaRII similarly reached a peak induction after 3–6 h of EGF treatment (Fig. 6C). Although with a weaker magnitude, a similar rapid induction in the levels of both endogenous and exogenously expressed TbetaRII mRNA occurred by EGF in the PC-3, but not the DU-145, androgen receptor-negative human prostate cancer cell lines (supplemental Fig. S1).


Figure 7
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  		FIGURE 7.
TGF-beta suppresses the expression of TbetaRII protein and mRNA in an EGF-reversible manner. LNCaP cells were infected in 1% DC-FBS + DMEM/F12 with a recombinant adenovirus expressing wild-type TbetaRII (AdMax-WT-TbetaRII) (1:200) for 24 h. Medium was replaced (without virus) before various times of treatment with 10 ng/ml of TGF-beta1 alone (A, B) or by pretreatment with 20 ng/ml of EGF (C, D), and cells were harvested for assaying TbetaRII by Western blot (A, C) or Northern blot (B, D) analysis. For co-treatment with EGF and TGF-beta, EGF was added 0.5 to 24 h prior to TGF-beta1. In the above time course experiments, the time of TGF-beta1 addition was varied, with total culture times and all other conditions kept constant for proper control. All data are representative of three independent experiments. Deglycosylated protein (20 µg) and total RNA (10 µg) were subjected to Western blot (A, C) or Northern blot (B, D) analyses, respectively. A representative blot of three independent experiments is shown.

 
TGF-beta1 Down-regulates mRNA and Protein Levels of TbetaRII: an EGF-reversible Mechanism—Interestingly, expression of TbetaRII at both protein and mRNA levels decreased as early as 3 h following TGF-beta1 addition, and a 30-min pretreatment of cells with EGF abrogated the TGF-beta1-induced loss of TbetaRII protein levels (Fig. 7, A–C). Similarly, EGF enhanced TbetaRII mRNA expression and reversed TGF-beta1 down-regulation of TbetaRII mRNA level following treatment of these cells with EGF for 24 h and then with TGF-beta1 for 24–48 h (Fig. 7D). Western blots done in parallel revealed similar changes in TbetaRII protein levels (supplemental Fig. S2B). To confirm that such expression is not a reflection of changes in CMV promoter activity, we showed that neither EGF nor TGF-beta1 altered other CMV-driven constructs (i.e. AdMax-Akt (supplemental Fig. S2B) or CMV-Renilla (data not shown)); the overexpression of Akt also did not change the expression of TbetaRII in the presence or absence of EGF or/and TGF-beta1 (data not shown). Taken together, these results suggest that EGF and TGF-beta1 enhances and suppresses, respectively, TbetaRII mRNA levels through changes in mRNA stability rather than in transcription.


Figure 8
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  		FIGURE 8.
EGF stabilizes TbetaRII mRNA through a mechanism that is independent of de novo transcription and protein synthesis. A, LNCaP cells were infected with AdMax-WT-TbetaRII (1:200) for 24 h and treated with actinomycin D (1 µg/ml) for 1 h, followed by treatment for the indicated times with EGF (20 ng/ml) or vehicle. All cultures were extracted for RNA at 8 h of the first EGF treatment time; 10 µg of total RNA was subjected to Northern blot analysis. Expression of TbetaRII mRNA detected by phosphorimaging was quantified by ImageQuant. B and C, effects of cycloheximide (1 mg/ml) on changes in TbetaRII mRNA expression following a 3-h treatment with 20 ng/ml of EGF (B) or 10 ng/ml of TGF-beta (C) were studied by Northern blot analysis. A representative blot of three independent experiments is shown.

 
Elevation of TbetaRII Levels by EGF Are Independent of de Novo Transcription and Protein Synthesis—The rapidity of the changes in TbetaRII mRNA levels by EGF or TGF-beta1 suggests that such regulation may not require de novo transcription or protein synthesis. To test these possibilities, we first measured changes in TbetaRII mRNA levels by EGF in the presence of a potent transcriptional inhibitor, actinomycin D. For this, cells overexpressing TbetaRII via AdMax-TbetaRII were pretreated with 1 µg/ml of actinomycin D for 1 h and then incubated for the indicated times in the presence or absence of EGF, followed by Northern blot analysis (Fig. 8A). ImageQuant analysis shows that TbetaRII mRNA was stabilized after 4 h of EGF treatment, with maximum protection by 6 h. We next determined whether de novo protein synthesis was necessary for these changes in the stability of TbetaRII. For this, cells were pretreated with 1 µg/ml of cycloheximide for 1 h before a 3-h treatment with EGF or TGF-beta1. In this experiment, cycloheximide neither blocked nor attenuated the effects of EGF or TGF-beta1 on exogenously expressed TbetaRII mRNA but actually enhanced all levels of TbetaRII mRNA (Fig. 8, B and C). Therefore, changes in TbetaRII stability by EGF or TGF-beta1donot require de novo protein synthesis.

Modulation of TbetaRII Levels by TGF-beta1 or EGF Does Not Require TbetaRII Kinase or Smad3—To understand the mechanism by which TGF-beta1 or EGF is able to modulate stabilization of TbetaRII, we next investigated whether such stabilization required autophosphorylation of TbetaRII or the kinase activity of this receptor. LNCaP cells were transiently transfected with expression constructs of TbetaRII mutated at the kinase domain or prominent sites of autophosphorylation (K277R, S213A, S409A, S416A, and S409A/S416A). Changes in expression of TbetaRII in these cells after 24 h of treatment with EGF or/and TGF-beta1 were then measured by Western blot analysis. Our data clearly show that TGF-beta1 treatment suppressed the level of each of the mutant receptors, similar to that of wild-type TbetaRII. On the other hand, EGF both enhanced the expression of these receptor mutants and reversed their down-regulation by TGF-beta1 (Fig. 9A). These data suggest that the kinase activity or autophosphorylation of TbetaRII is not required for stabilization of TbetaRII by EGF or destabilization by TGF-beta1 despite major differences in the ability of these receptors to mediate induction of AP-1-luc activity by TGF-beta1 (supplemental Fig. S3).

To explore the requirement of Smad3 on TGF-beta1- and EGF-mediated changes in TbetaRII levels, we silenced Smad3 expression in LNCaP cells with siRNA oligonucleotides (si-Smad3) (Fig. 9B). Loss of Smad3 by si-Smad3 blocked the ability of TGF-beta1 to activate AP-1-luciferase (supplemental Fig. S4) but did not block the ability of EGF to enhance levels of TbetaRII. However, si-Smad3 significantly elevated TbetaRII in the non-treated control and, to a smaller extent, in the TGF-beta-only-treated group (Fig. 9B). Moreover, si-Smad3 did not enhance the levels of TbetaRII over that stabilized by EGF alone or by EGF + TGF-beta1 (Fig. 9B). These data thus support that the changes in TbetaRII expression by EGF or TGF-beta1 occur through a Smad3-independent mechanism(s). Moreover, these data also suggest that Smad3 may down-regulate the levels of TbetaRII and reduce the observed effectiveness of EGF to stabilize TbetaRII levels.

Another interesting observation from Fig. 9B was that expression of Smad3 in the control (scrambled siRNA) group was enhanced by TGF-beta1 and further elevated by the inclusion of EGF, but not by EGF without TGF-beta1. The latter data suggest that Smad3 is under positive regulatory control by TGF-beta1 in these cells. Although elevated expression of Smad3 by TGF-beta1 may contribute to ligand-dependent loss of TbetaRII, the ability of TGF-beta1 to reduce levels of TbetaRII in the absence of protein synthesis (Fig. 8C) further supports the involvement of a Smad3-independent mechanism for enhanced destabilization of TbetaRII by TGF-beta1.

Stabilization of TbetaRII by EGF Is EGFR Kinase-dependent and Occurs through a MEK1/2-dependent Pathway—Autophosphorylation of activated EGFR stimulates a number of intracellular signal transduction cascades, including the Ras/Raf/mitogen-activated protein kinase and PI3-kinase/Akt pathways (56–58). To better understand how EGF stabilizes TbetaRII expression, we first investigated the kinase dependence of EGFR on regulating TbetaRII levels, using specific inhibitors of EGFR kinase, PD153035 or AG1478. Pretreatment with each of these inhibitors suppressed stabilization of TbetaRII by EGF either in the presence or absence of TGF-beta1 (Fig. 10, A and B), suggesting that EGFR kinase activity is necessary for downstream pathways involved in stabilization of TbetaRII. We next defined these downstream pathways using selective kinase inhibitors, including U0126 (MEK1/2), PD98059 (MEK1), SB202190 (p38K), LY294002 (PI3-K), rapamycin (mTOR), and SP600125 (SAPK/c-Jun N-terminal kinase). Of these, only U0126 abolished EGF-induced TbetaRII stabilization (Fig. 10, C and D). These inhibitors were functionally tested by their ability to block phosphorylation of downstream substrates such as p44/42 mitogen-activated protein kinase, p70S6K, and c-Jun (Fig. 10, C and D). PD98059 also inhibited the up-regulation of TbetaRII by EGF, whereas the other agents were ineffective in this respect, thus implicating that MEK1/2 is the mechanism by which EGF enhances TbetaRII levels.


Figure 9
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  		FIGURE 9.
The ability of EGF and TGF-beta to modulate levels of TbetaRII is independent of the phosphorylation status or kinase activity of TbetaRII or the expression of Smad3. A, LNCaP cells were transfected with kinase-dead (K277R) and autophosphorylation (S213A, S409A, S416A, or S409A/S416A) mutants of TbetaRII (48), followed by incubation with or without EGF (20 ng/ml). 16 h later cells were treated with vehicle or TGF-beta1 (10 ng/ml) for an additional 24 h and then analyzed for TbetaRII expression by Western blot. B, separately, cells were co-transfected with TbetaRII and either 80 nM Smad3 siRNA or scrambled siRNA for 4 h, followed by treatment with or without EGF (20 ng/ml). 16 h later, cells were incubated in the absence or presence of TGF-beta1 (10 ng/ml) for an additional 24 h. For Western blot analysis of TbetaRII, 20-µg aliquots of protein lysates were deglycosylated prior to electrophoresis (A, B). A representative blot of three independent experiments is shown.

 


Figure 10
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  		FIGURE 10.
Modulation of TbetaRII levels by EGF occurs through an EGFR kinase-dependent and MEK1/2-kinase-dependent pathway. LNCaP cells transiently overexpressing TbetaRII were pretreated with PD153035 (5 µM)(A); AG1478 (200 nM) (B); rapamycin (R, 200 nM), SB202190 (SB, 10 µM), U0126 (U, 10 µM) (C); or PD98059 (PD, 10 µM), LY294002 (LY, 10 µM), or SP600125 (SP, 10 µM)(D) 1–2 h prior to addition of EGF (A–D) and then incubated with TGF-beta1 for 24 h. E, overexpression of MEK1 enhanced exogenous TbetaRII mRNA levels, and suppression of MEK1/2 activity by U0126 abolished the EGF-mediated stabilization of TbetaRII mRNA levels. F, effects of U0126 and AG1478 (1 h pretreatment) on induction of endogenous TbetaRII mRNA by EGF (3 h) in LNCaP cells were assayed by RT-PCR as described in Fig. 3C. A representative blot of three independent experiments is shown (A–F).

 
We further examined whether overexpression of MEK1 could enhance TbetaRII mRNA, mimicking this EGF effect, and whether U0126 and AG1478 could also suppress TbetaRII mRNA stabilized by EGF, using Northern blot and RT-PCR analyses (Fig. 10, E and F). Consistent with our Western blot data, TbetaRII mRNA was significantly elevated by enforced expression of MEK1, and U0126 completely abolished the EGF effect on TbetaRII mRNA levels (Fig. 10E). Furthermore, U0126 and AG1478 reversed the ability of EGF to enhance the expression of endogenous TbetaRII mRNA in LNCaP cells not transfected or infected with TbetaRII (Fig. 10F). As expected, AP-1-luc activity permissively induced by TGF-beta1 + EGF was depressed 9- and 16-fold, respectively, by the MEK1/2 inhibitors PD98059 and U0126 (supplemental Fig. S5). Consistently, the p38-kinase inhibitor (SB), which was not able to block TbetaRII stabilization by EGF, also failed to block AP-1 reporter activity (supplemental Fig. S5).

Consistent with the above results, enforced expression of MEK1 (≥50 ng of the vector) stabilized TbetaRII levels (supplemental Fig. S6A), and overexpression of active H-Ras and c-Fos, but not Egr-1 or Elk-1, also significantly increased TbetaRII levels (supplemental Fig. S6B). These results further support that such changes in levels of TbetaRII occur through a MEK1/2-dependent pathway and may also involve the activation of c-Fos but not Egr-1 or Elk-1. Taken together, these data support that EGF permits the transduction of TGF-beta1 signals mainly through TbetaRII mRNA stabilization via an EGFR-Ras-MEK1/2-dependent, but Smad3-independent, mechanism.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have provided the first evidence supporting a novel function of EGF as a permissive factor for numerous TGF-beta responses, including growth suppression. Our data demonstrate that EGF targets TbetaRII through a previously unreported mechanism, namely via enhanced stabilization of TbetaRII message. Moreover, this is the first report that TGF-beta down-regulates TbetaRII mRNA by a non-transcriptional mechanism. Such ligand-mediated receptor down-regulation occurs similarly with either wild-type TbetaRII or kinase-dead TbetaRII and is reversed by pretreatment with EGF, but not by silencing Smad3 with siRNA. These results suggest that TGF-beta destabilizes TbetaRII mRNA through a non-classical TGF-beta signaling pathway that appears to be reversed by EGF. Our results can be distinguished from previous reports of ligand-dependent loss of TbetaRII protein occurring through receptor-mediated endocytosis (59), which is reported to be required for the transduction of TGF-beta signals (60).

Upon activation of EGFR by ligand binding and receptor dimerization, EGF initiates the recruitment and phosphorylation of several intracellular substrates, activating multiple signaling cascades such as PI3-kinase, STAT, Ras/MEK, and Rac/PAK/c-Jun N-terminal kinase (26). Using various kinase inhibitors we have shown that EGF controls TbetaRII levels in LNCaP cells through an EGFR kinase-dependent mechanism that involves activation of MEK1 but not PI3-kinase, c-Jun N-terminal kinase, mTOR, or p38-kinase. As expected, Ras or MEK1 cDNA expression constructs enhanced TbetaRII levels. c-Fos, but not Egr-1, both of which are induced by EGF (Fig. 1D), may be involved in the stabilization of TbetaRII by EGF, as we showed that enforced expression of c-Fos but not Egr-1 induces TbetaRII expression (supplemental Fig. S6B). Therefore, our results indicate that an EGFR kinase/Ras/MEK1-dependent pathway mediates the permissive action of EGF on TGF-beta signaling. Further data suggest that c-Fos, but not Egr-1 or Elk-1, is a potential downstream mediator of this EGF activity, although additional work awaits identification of the actual mediator(s).

The fact that the TbetaRII expression constructs used here are devoid of the TbetaRII untranslated regions suggests that regulatory elements for stabilization by EGF are located within the coding region of TbetaRII rather than within its untranslated regions. This is similar to stabilization of c-Fos, c-Myc, and beta-tubulin mRNAs, which are also controlled by sequences within their coding region (61). Specific developmentally controlled proteins have been identified that bind to discreet coding regions of c-Myc and c-Fos mRNAs and regulate their message half-lives (61, 62). One of these coding region instability determinant-binding proteins, whose expression is greatly elevated in ~30% of human breast cancers, has recently been reported to cause mammary tumors in transgenic mice when targeted to the mammary gland with a WAP promoter construct (63). Our data on TbetaRII constructs harboring mutations at kinase and autophosphorylation sites (K277R, S213A, S409A, S416A, and S409A/S416A) indicate that these sites are not involved in TbetaRII down-regulation by TGF-beta or reversal of such down-regulation by EGF. We are currently exploring the sites of TbetaRII involved in such mRNA loss, using various deletion constructs of the TbetaRII coding sequence to help identify potential coding region-binding proteins for TbetaRII. The results of our cycloheximide experiments suggest that the EGF or TGF-beta controls of such putative coding region-binding protein(s) involve post-translation modification rather than de novo protein synthesis. Identification of such proteins may have therapeutic potential in reversing changes in TbetaRII levels occurring during carcinogenesis.

Our proposed role of EGF as a stabilizer of TbetaRII adds a new perspective on the mechanism of cross-talk between EGF and TGF-beta. EGF has previously been reported to suppress TGF-beta responses in other epithelial cells (30, 64) or be permissive to TGF-beta responses in fibroblasts. Particularly striking is the synergism of EGF and TGF-beta for growth of NRK-49F rat kidney fibroblast cells on monolayer cultures (35) and in soft agar (65, 66). TGF-beta has been suggested to promote EGF responses by inducing levels of EGFR in the NRK-49F cell line (67). We recently found that EGF enhances TbetaRII expression and TGF-beta decreases levels of this receptor in NRK-49F cells (data not shown), similar to LNCaP cells. However, unlike in LNCaP cells, in NRK-49F cells EGF only partially reverses the loss of TbetaRII expression by TGF-beta1. NRK-49F cells may behave similarly to human dermal fibroblasts, in which EGF was shown to activate the TbetaRII promoter through a PI3-kinase-dependent mechanism (68).

EGF has previously been reported to also suppress TGF-beta signaling in a variety of cell lines not used in our current study. These may occur through EGF-induced 1) inactivation of Smad2 by phosphorylation of its middle linker region (69), 2) activation of a transcriptional repressor (TIGF) through Ras (30), or 3) inhibition of downstream signals of TGF-beta-induced apoptosis (such as caspase-9 activation) via a PI3-kinase-dependent pathway (31). Taken together, these studies suggest that the manner by which EGF affects TGF-beta responses is cell type and context dependent. Our data showing a permissive effect of EGF on TGF-beta were obtained using the most widely studied androgen-dependent human adenocarcinoma cell line, LNCaP. Although isolated from a lymph node metastasis, LNCaP cells appear to represent a well differentiated and androgen-responsive prostate carcinoma with neuroendocrine phenotype (70). However, such regulation of TbetaRII found in LNCaP cells may not be specific to prostate carcinomas with neuroendocrine phenotype, because we showed similar (although reduced in magnitude) responses on the PC-3 human prostate carcinoma cell line, which lacks neuroendocrine behavior.

In conclusion, we propose a new model for control of TGF-beta responses by EGF. In our model, EGF may enhance or permit TGF-beta responses, such as growth suppression, through TbetaRII mRNA stabilization controlled by its coding sequence. Our results suggest that the activation of EGFR in certain cancers may function to prevent full loss of TbetaRII expression in late stage cancers and thereby permit some of the direct oncogenic behavior of TGF-beta acquired during tumor progression (25). Moreover, our results suggest that the therapeutic use of EGFR inhibitors for early stage cancer may impede on therapy by relieving the tumor-suppressive effects of TGF-beta through decreasing the stability of TbetaRII. Further work remains to identify targets downstream of EGFR, MEK1/2 or c-Fos that control TbetaRII levels and the TbetaRII coding region-binding protein(s) involved in such regulation. This effort may provide new insight on the regulation of TbetaRII expression and disclose novel therapeutic strategies to modulate responses of TGF-beta during carcinogenesis.


   FOOTNOTES
 
* This work was supported by NCI, National Institutes of Health Grants R01CA092102 and R01CA102074. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S6. Back

1 To whom correspondence should be addressed: Case Cancer Center Research Laboratories, Wolstein Research Bldg., Rm. 3532, 2103 Cornell Rd., Cleveland, OH 44106. Tel.: 216-368-5670; Fax: 216-368-8919; E-mail: dxd49{at}case.edu.

2 The abbreviations used are: TGF-beta, transforming growth factor-beta; TbetaRI, TGF-beta type I receptor; TbetaRII, TGF-beta type II receptor; AP-1, activator protein-1; CMV, cytomegalovirus; DC, dextran-coated charcoal-treated; DMEM/F12, Dulbecco's modified Eagle's medium/Ham's F12; EGF, epidermal growth factor; EGFR, EGF receptor; FBS, fetal bovine serum; PI3-kinase, phosphatidylinositol 3-kinase; RT, reverse transcription; siRNA, small interfering RNA; WT, wild-type; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Joan Massagué for 3TP-lux, Dr. Xiao-Fan Wang for p21WAF-1/CIP1 promoter-luc, Dr. Harvey Lodish for TbetaRII mutant constructs (S213A, S416A, S409A/S416A), Dr. Jeffrey L. Wrana for wt-TbetaRII and TbetaRI (ALK5), Dr. Seong-Jin Kim for TbetaRII promoter-Luc constructs (–1690/+38, –216/+35), Dr. Binwu Tang and Dr. Lalage Wakefield for the dominant-negative-TbetaRII construct, Dr. Aris Moustakas for help with designing the siRNA sequences, Dr. Anthony Zeleznik for the adenoviral protocol, and Susan Cornelius for technical assistance.



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