Nerve growth factor mediates activation of the Smad pathway in PC12 cells Marion Lutz 1 , Kerstin Krieglstein 2 , Simone Schmitt 1 , Peter ten Dijke 3 , Walter Sebald 1 , Andrea Wizenmann 4 and Petra Knaus 1 1 Department of Physiological Chemistry II, Biocenter, University of Wu ¨ rzburg, Germany; 2 Department of Anatomy, University of Go ¨ ttingen, Germany; 3 Division of Cellular Biochemistry, the Netherlands Cancer Institute, Amsterdam, the Netherlands; 4 JRG (of) Developmental Neurobiology, Biocenter, University of Wu ¨ rzburg, Germany Ligand-induced oligomerization of receptors is a key step in initiating growth factor signaling. Nevertheless, complex biological responses often require additional trans-signaling mechanisms involving two or more signaling cascades. For cells of neuronal origin, it was shown that neurotrophic effects evoked by nerve growth factor or other neurotro- phins depend highly on the cooperativity with cytokines that belong to the transforming growth factor b (TGF-b) superfamily. We found that rat pheochromocytoma cells, which represent a model system for neuronal differentiation, are unresponsive to TGF-b1 due to limiting levels of its receptor, TbRII. However, stimulation with nerve growth factor leads to activation of the Smad pathway independent of TGF-b. In contrast to TGF-b signaling, activation of Smad3 by nerve growth factor does not occur via phospho- rylation of the C-terminal SSXS-motif, but leads to hetero- meric complex formation with Smad4, nuclear translocation of Smad3 and transcriptional activation of Smad-dependent reporter genes. This response is direct and does not require de novo protein synthesis, as shown by cycloheximide treat- ment. This initiation of transcription is dependent on func- tional tyrosine kinase receptors and can be blocked by Smad7. These data provide further evidence that the Smad proteins are not exclusively activated by the classical TGF-b triggered mechanism. The potential of NGF to activate the Smad pathway independent of TGF-b represents an import- ant regulatory mechanism with special relevance for the development and function of neuronal cells or of other NGF- sensitive cells, in particular those that are TGF-b-resistant. Keywords:PC12cells;Smads;crosstalk;nervegrowth factor; transforming growth factor-b. Proteins of the transforming growth factor b (TGF-b) family are multifunctional cytokines that display a very broad range of biological activities including cell prolifer- ation, differentiation and apoptosis [1]. TGF-bs are ubi- quitously expressed and act on virtually all tissues, thereby causing distinct cell-specific effects depending on the present composition of receptors, Smad proteins and DNA-binding partners [2,3]. Referring to cell populations of neuronal origin, TGF-bs are described to possess neurotrophic effects when acting in concert with other cytokines or neurotro- phins [4,5]. Signals mediated by TGF-b are propagated by two receptor serine/threonine kinases designated as TGF-b type I (TbRI) and type II (TbRII) receptors [6,7]. The type II receptors comprise TbRII [8] and its alternative splice variant TbRII-B [9]. The initial binding of TGF-b1to TbRII is followed by recruitment and activation of TbRI [10]. Receptor-associated Smads (R-Smads) involved in TGF-b signaling (Smad2 and Smad3) are phosphorylated at the C-terminal SSXS-motif [11,12], interact with the common mediator Smad4 [13] and translocate to the nucleus to mediate specific transcriptional responses [14,15]. Although Smad2 and Smad3 share 92% amino acid identity, they are functionally distinct. A short amino acid sequence in the MAD homology 1 (MH1) domain of Smad2 is responsible for its inability to bind DNA [16,17]. However, Smad3 can directly bind to a specific DNA sequence termed the Smad binding element (SBE). These distinct properties account for activation of different subsets of target genes by either Smad2 or Smad3. Various proteins have been identified that negatively influence TGF-b signaling at different levels [18]. One of these proteins, the inhibitory Smad7, mediates its antagon- istic effects by stable interaction with TbRI, thus preventing the transient contact of R-Smads with the receptor and blocking the proceeding cascade [19,20]. In addition, Smad7 was shown to recruit the E3 ubiquitin ligases Smurf1 and Smurf2 to TbRI, thereby triggering degradation of the TGF-b receptor complex [21,22]. Interestingly, expression of Smad7 is rapidly induced in response to TGF-b1[23]and therefore plays a crucial role in regulating TGF-b signaling. Correspondence to P. Knaus, Physiological Chemistry II, Biocenter, University of Wu ¨ rzburg, 97074 Wu ¨ rzburg, Germany. Fax: + 49 931 888 4113, Tel.: + 49 931 888 4127, E-mail: pknaus@biozentrum.uni-wuerzburg.de Abbreviations:TGF-b, transforming growth factor-b;TbR, TGF-b receptor type; NGF, nerve growth factor; R-Smads, receptor-associ- ated Smads; MH, Mad homology domain; SBE, Smad binding element; TrkA, tyrosine kinase receptor; ECD, extracellular domain; MAPK, mitogen activated protein kinase; RSK, receptor serine/ threonine kinase; BMP, bone morphogenetic protein; GFP, green fluorescent protein. (Received 5 November 2003, accepted 15 January 2004) Eur. J. Biochem. 271, 920–931 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03994.x In contrast to TGF-b signaling through receptor serine/ threonine kinases, nerve growth factor (NGF) signals via a tyrosine kinase receptor (TrkA) which initiates multiple pathways, the most prominent being the Ras/Raf-MAPK pathway [24,25]. Besides TrkA, NGF can also bind to a low-affinity neurotrophin receptor, p75 NTR ,whichisa member of the tumor necrosis factor cytokine receptor family [26]. It is increasingly evident that signal transduction in general does not only occur in a linear fashion but rather comprises a complex network of signaling pathways that mutually influence their activity [27]. TGF-b family members are implicated in multiple interdependent signals between pathways originating from receptor serine/threo- nine kinases and receptor tyrosine kinases. Cellular responses induced by bone morphogenetic protein (BMP) for example, can be impaired by epidermal growth factor and hepatocyte growth factor, which lead to the phosphorylation of Smad1 in the linker region and thus prevent Smad1 nuclear translocation [28]. Direct effects of signaling intermediates were shown for the c-Jun N-terminal kinase and protein kinase C. c-Jun N-terminal kinase phosphorylates Smad3 outside the SSXS-motif, thus supporting nuclear transport of Smad3 [29]. Protein kinase C however, abrogates direct DNA binding of Smad3 by serine phosphorylation in the MH1 domain [30]. This indicates that Smads are not restricted to TGF- b/BMP pathways; rather, they represent a point of convergence of various signals and their activation is a precise contextually regulated process. Here we demonstrate that in rat pheochromocytoma cells (PC12), NGF stimulation results in activation of the Smad cascade. This Smad activation is independent of TGF-b1 and occurs by a mechanism which is different from that induced by TGF-b1 in that it does not lead to C-terminal phosphorylation of R-Smads. However, NGF rapidly triggers association between Smad3 and Smad4, translocation to the nucleus and gene expression. NGF- mediated transcriptional activation of TGF-b responsive reporter constructs requires the presence of functional TrkA receptors and can be impaired by the inhibitory Smad7. Materials and methods Antibodies and reagents The monoclonal antibody against TGF-b1, -b2and-b3 (clone #1D11) was purchased from R&D Systems. Details of polyclonal antisera against Smad2 (anti-S2), Smad3 (anti-S3) and C-terminally phosphorylated forms of Smad1 (anti-PS1) and Smad2 (anti-PS2) were published previously [31,32]. The P-Smad1 antibody shows cross- reactivity to phosphorylated Smad3 and can thus be used as an Ôanti-P-Smad3Õ (anti-PS3) [33]. The monoclonal antibody Smad2/3 was purchased from BD Biosciences and the monoclonal antibody Smad4 (B-8) was obtained from Santa Cruz. Peroxidase-coupled goat anti-(rabbit IgG) Ig was obtained from Dianova. Doxycycline was purchased from Sigma. Human TGF-b1wasfromR&D Systems and mouse NGF (2.5S) from Alomone labs (Jerusalem, Israel). Coating of culture dishes with rat tail collagen Collagen was isolated from rat tails according to standard protocols. 1 Appropriate measures were taken to minimize animal pain and discomfort according to the European Communities Council Directive of 24 November 1986 (86/609/EEC). Culture dishes were incubated with a solution of 38 lgÆmL )1 collagen in 0.1% (v/v) acetic acid for at least 1 h followed by thorough washing with sterile dH 2 O and medium without supplements. Neutralization of TGF-b All TGF-b isoforms were neutralized by the addition of either monoclonal antibodies against TGF-b1, -b2, -b3 (20 lgÆmL )1 ) or a 100- or 1000-fold molar excess of the soluble extracellular domain (ECD) of TbRII-B (TbRII-B- ECD), kindly provided by J. Nickel (Biocenter, University of Wu ¨ rzburg, Germany). DNA constructs Smad2 and Smad7 constructs were published previously [20,34]. The Smad3 construct was kindly provided by R. Derynck (University of California at San Francisco, CA, USA). Smad1, Smad4 and Smad4 (DSAD) constructs were a gift from M. de Caestecker (National Cancer Institute, Bethesda, MD, USA) [35]. The NGF receptor constructs (TrkA and TrkA-K538R) were obtained from M. Chao (Skirball Institute of Biomolecular Medicine, New York, NY, USA). Cell culture and transient transfection PC12 cells [36] were cultured in RPMI supplemented with 10% (v/v) horse serum, 5% (v/v) fetal bovine serum and antibiotics 2 (penicillin, 100 UÆmL )1 and streptomycin, 100 lgÆmL )1 ). Transient transfection of PC12 cells was performed using LipofectAMINE TM (Life Technologies Inc.) according to the manufacturer’s protocol. 293T cells were maintained in minimum Eagle’s medium (MEM) containing 10% (v/v) fetal bovine serum and antibiotics. Transfection of 293T cells was performed using calcium phosphate–DNA coprecipitation [37]. L6 rat myoblasts were cultured in DMEM with 10% (v/v) fetal bovine serum. MLEC cells [38] were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum and 250 lgÆmL )1 geneticin. Retroviral constructs and transduction of PC12 cells by retroviral transfer The retroviral construct pMX-GFP-Smad3 was a kind gift from Y. Henis (Tel Aviv University, Israel) [39]. The construct for N-terminally HA-tagged TbRII-wt was subcloned into the retroviral vector pczCFG-EGIRT (D. Lindemann, unpublished results) 3 downstream of a tetracycline-inducible cytomegalovirus (CMV) minimal promoter [40]. Cells were transduced by infection with helper-free VSV-G pseudotyped retroviruses as described previously [37]. Briefly, 293T cells were cotransfected with the retroviral construct and plasmids for gag-pol and Ó FEBS 2004 Crosstalk between NGF and TGF-b pathways (Eur. J. Biochem. 271) 921 VSV-G. Twenty-four hours post-transfection, cells were treatedwith10m M sodium-butyrate for 10 h. Infection of the target cells was performed 48 h and 72 h after transfec- tion. Because the retroviral sequences contain the gfp gene, infected cells could be selected by FACS sorting. Luciferase reporter gene assays For reporter gene assays, different TGF-b responsive elements were used. The p3TP-luc(+) reporter is derived from the p3TP-luc construct [7] but contains a modified luc gene (pSP-luc(+), Promega). The pSBE reporter [41] serves as a readout for TGF-b as well as BMP signaling whereas p3TP-luc(+) and (CAGA) 12 -luc constructs respond speci- fically to TGF-b mediated signals [42,43]. PC12 cells were plated on collagen-coated 6-well plates prior to cotransfection with reporter constructs, pRL-TK (Renilla luciferase, Promega) and with the indicated receptor or Smad constructs. The total amount of DNA was kept constant by the addition of empty vector (pcDNA3). Twenty-four hours post-transfection, cells were starved in medium containing 0.2% (v/v) horse serum for 4 h, followed by stimulation with either 2 n M NGF or 200 p M TGF-b1 for an additional 24 h. Cell lysis and luciferase measurement were performed according to manufacturer’s instructions (Dual Luciferase Assay system, Promega) and data were normalized using Renilla luminescence. The value of unstimulated transfected cells was set to one and all other values were calculated accordingly. Smad-Western blotting To investigate Smad phosphorylation, cells were seeded on Petri dishes, starved for 4 h in medium containing 0.2% (v/v) serum and stimulated with 2 n M NGF or 200 p M TGF-b1 for 30 min. Cells were lysed in TNE lysis-buffer [20 m M Tris/HCl pH 7.4, 150 m M NaCl, 1% (v/v) Triton X-100, 1 m M EDTA, 1 m M phenylmethanesulfonyl fluor- ide] containing protease inhibitors (Complete TM , Roche) and phosphatase inhibitors (50 m M NaF, 10 m M Na 4 P 2 O 7 and 1 m M Na 3 VO 4 ). Equal amounts of cell lysates were analyzed by immunoblotting using anti-PS2, anti-Smad2, anti-PS3 or anti-Smad3. Immunoreactive proteins were visualized by enhanced chemiluminescence 4 . Co-immunoprecipitation of Smad proteins PC12 cells (5 · 10 6 ) were starved in medium containing 0.2% (v/v) horse serum for 4 h. Ligand stimulation was carried out for the indicated periods of time using 2 n M NGF. Cells were lysed in a buffer containing NaCl/P i pH 7.4, 0.5% (v/v) Triton X-100, 1 m M EDTA, phospha- tase inhibitors and protease inhibitors. Cell lysates were incubated with monoclonal antibody Smad2/3 or Smad4 for 2 h, followed by incubation with protein-A sepharose beads overnight at 4 °C. The beads were washed twice with lysis buffer and twice with NaCl/P i before immunocom- plexes were eluted by boiling in SDS sample buffer for 5 min. Following separation by SDS/PAGE and electro- transfer to a nitrocellulose membrane, proteins were immunoblotted with Smad4 or Smad2/3 antibodies as appropriate. Nuclear and cytoplasmic fractionation PC12 cells and L6 cells were starved in low serum medium for 4 h. Ligand stimulation was performed for the indicated periods of time with 2 n M NGF or 200 p M TGF-b1, respectively. Control cells were stimulated with 2 n M NGF for 1 h following a 1 h treatment with cycloheximide (5 lgÆmL )1 in dimethylsulfoxide) or dimethylsulfoxide only. Cells were washed with NaCl/P i , centrifuged (1000 g,4°C, 10min) and the cell pellet was then resuspended in hypotonic buffer (10 m M Hepes pH 7.9, 1.5 m M MgCl 2 ,10m M KCl, protease inhibitors). Cells were vortexed thoroughly and cell lysis was followed by microscopy until 90% of the cells were lysed. Following centrifugation (1000 g,4°C, 10 min), the supernatant was referred to as the cytoplasmic fraction. The pellet containing the nuclei was resuspended in high salt buffer [20 m M Hepes pH 7.9, 25% (v/v) glycerol, 420 m M NaCl 2 ,1.5m M MgCl 2 ,0.2m M EDTA, protease inhibitors]. Extraction of nuclear proteins was achieved by vortexing this solution thoroughly, incubating for 30 min on ice and subsequent centrifugation (25 000 g,4°C, 20 min). The supernatant was collected and represents the nuclear fraction. Nuclear translocation PC12 cells were plated on collagen-coated dishes. Starvation in a low serum medium for 4 h was followed by stimulation with 2 n M NGF for the indicated times. Control cells were stimulated with 2 n M NGF for 30 min following a 1 h treatment with cycloheximide (5 lgÆmL )1 in dimethylsulf- oxide) or dimethylsulfoxide only. The cells were then washed with NaCl/P i and 3% (v/v) BSA, fixed in 4% (v/v) paraformaldehyde and 0.2% (v/v) TX-100 for 10 min at room temperature. After washing with NaCl/P i containing 3% (v/v) BSA, Smad2/3 staining was performed with antibodies from BD Biosciences. Nuclei were stained by the addition of 1 lgÆmL )1 Hoechst 33342 for 2 min. The subcellular distribution of Smad2/3 was then analyzed by confocal microscopy. Results NGF mediates the activation of Smad-dependent reporter genes independently of TGF-b Survival of neuronal cells is described to be synergistically promoted by TGF-bs and neurotrophic factors (e.g. NGF) [44,45]. To examine whether NGF has the potential to modulate the Smad pathway in PC12 cells, we performed reporter gene assays using luciferase constructs containing promoter elements that are responsive to proteins of the TGF-b superfamily, i.e. pSBE-luc, p3TP-luc(+) and (CAGA) 12 -luc [7,41,42]. As indicated in Fig. 1A, PC12 cells show a significant increase of transcriptional activation after stimulation with NGF on all tested Smad-dependent reporter constructs. In contrast, TGF-b1 is not able to induce transcription from these reporters in PC12 cells. Different approaches were chosen to exclude that this NGF- mediated transcriptional response is a secondary effect, caused for instance by NGF-triggered secretion of TGF-b1 as published previously [46]. First, the reporter gene assay 922 M. Lutz et al.(Eur. J. Biochem. 271) Ó FEBS 2004 was carried out in the presence of monoclonal antibodies against TGF-b1, -b2and-b3 to neutralize all TGF-b isoforms (Fig. 1B). Stimulation with NGF leads to a significant increase in transcriptional activity which is not impaired by the presence of neutralizing TGF-b antibodies. TGF-b, however, does not induce luciferase activity, neither Fig. 1. NGF mediates transcription from TGF-b responsive reporter genes. (A) PC12 cells were plated on collagen-coated dishes and cotransfected with pRL-TK and pSBE-luc, p3TP-luc(+) or (CAGA) 12 -luc. Following starvation for 4 h in medium containing 0.2% (v/v) horse serum, cells were stimulated for 24 h with either 2 n M (50 ngÆmL )1 )NGF (black bars) or 200 p M TGF-b1(graybars),or were left untreated (white bars). Cell lysates were prepared and the luciferase activity was measured. Data were normalized as described in Materials and methods, and error bars represent the SD evaluated from three inde- pendent experiments. (B) Transfection and starvation of PC12 cells was carried out as described in (A) using pSBE-luc as the repor- ter construct. Subsequently, cells were treated with 2 n M NGF (black bars) or 200 p M TGF- b1 (gray bars) either in presence or in absence of 20 lgÆmL )1 TGF-b1, -b2, -b3antibody. After 24 h, luciferase activity was recorded and the data were evaluated as described above. (C) L6 cells stably expressing GFP-Smad3 were starved for 4 h followed by treatment with either 20 lgÆmL )1 anti- (TGF-b1, -b2, -b3) (lane 2), 200 p M TGF-b1 (lane 3) or 200 p M TGF-b1 together with 20 lgÆmL )1 anti-(TGF-b1, -b2, -b3) (lane 4). Cell lysates were analysed for C-terminally phosphorylated Smad3 (upper panel) or for total amounts of Smad3 (lower panel). (D) PC12 cells were cotransfected with pSBE and pRL-TK. The day after transfection, PC12 cells were starved as described previously and stimulated with either 2 n M NGF (black bars), 200 p M TGF-b1 (gray bars) or were left untreated (white bars). In addition, the experiment was carried out in the absence or presence of the soluble extracellular domain of TbRII-B (TbRII-B-ECD). TbRII-B-ECD was used at 100-fold and 1000-fold molar excess as indicated. Twenty-four hours after stimulation, the supernatant of the PC12 cells was transferred to L6 cells (lower panel) which were equivalently transfected and starved prior to addition of the supernatant. PC12 cells (upper panel) as well as L6 cells (lower panel) were lysed and tested for luciferase activity. Error bars represent the SD from two independent experiments. Ó FEBS 2004 Crosstalk between NGF and TGF-b pathways (Eur. J. Biochem. 271) 923 in the presence nor in the absence of antibodies. The neutralizing capacity of the TGF-b1, -b2and-b3 antibodies was verified in L6 cells stably transduced with a GFP– Smad3 construct (Fig. 1C). In the absence of TGF-b1, -b2 and -b3 antibodies, stimulation with TGF-b1leadsto C-terminal phosphorylation of Smad3, whereas treatment with TGF-b1 together with neutralizing antibodies impedes the phosphorylation of Smad3. Second, we performed a complementary experiment using the soluble ECD of TbRII-B, a TGF-b type II receptor splice variant that binds all three TGF-b isoforms [9]. TbRII-B-ECD was added in two different concentrations (100- and 1000-fold molar excess) to PC12 cells transfected with the pSBE-luc reporter in the presence of NGF or TGF-b1(Fig.1D, upper panel). The TbRII-B-ECD did not abolish NGF- mediated Smad activation in PC12 cells. Next, we harvested the supernatant of PC12 cells treated in this way and placed it on the TGF-b sensitive L6 myoblast cell line that was equally transfected with pSBE-luc (Fig. 1D, lower panel). As there was no detectable increase in luciferase activity in TGF-b-sensitive L6 cells that were treated with the super- natant from NGF stimulated PC12 cells (Fig. 1D, lower panel, bar 2), we can conclude that NGF treatment of PC12 cells does not lead to the production of active TGF-b.In contrast, the TGF-b-treated PC12 cell supernatant results in reporter activation in L6 cells (Fig. 1D, lower panel, bar 3). This activation is almost completely blocked by a 1000-fold molar excess of TbRII-B-ECD (Fig. 1D, lower panel, bar 9). Furthermore, we measured the amount of TGF-b that is produced in response to NGF and checked whether this TGF-b is present in an active or latent form. The quantification of TGF-b was performed by using the MLEC cell line, which stably expresses a luciferase reporter gene under the control of a truncated PAI-1 promoter [38]. We found that NGF leads to production of only marginal amounts ( 1.1 p M )ofactiveTGF-b (data not shown). This is in accordance with the results presented above (Fig. 1D, lower panel). Taken together, all approaches clearly demonstrate that NGF mediates the activation of the Smad pathway independently of the TGF-b ligand. TGF-b resistance is due to limiting amounts of TbRII in PC12 cells The expression of TGF-b receptors, particularly TbRII, in PC12 cells is controversially discussed in the literature [47,48]. Phosphorylation studies and reporter gene assays demonstrate that TbRII represents the limiting component of TGF-b1 signaling in PC12 cells. Smad2 phosphorylation wasanalysedinparentalPC12cellsandinstablePC12cells expressing TbRII-wt under the control of a doxycycline- inducible promoter. Figure 2A shows that treatment with TGF-b1 results in C-terminal phosphorylation only in PC12 cells that ectopically express TbRII but not in parental PC12 cells. However, in response to NGF, there is no phosphorylation of Smad2 at the SSXS-motif. Luciferase assays also show that transient transfection of PC12 cells with TbRII but not TbRI constructs leads to increased responsiveness to TGF-b1 (Fig. 2B), again indi- cating that TbRII is the limiting component of TGF-b1 signaling in PC12 cells. Mechanism of Smad activation by NGF The mechanism of Smad reporter activation was investigated by using luciferase constructs that allowed us to distinguish between signals originating from different R-Smads, i.e. pSBE-luc, p3TP(+)-luc and (CAGA) 12 -luc [7,41,42] (see Materials and methods). Using p3TP-luc(+), NGF-induced reporter activation was investigated after ectopic expression of various R-Smad constructs (Fig. 3A). From all R-Smads tested, Smad3 shows the most prominent induction of transcription after NGF stimulation. Similar results were obtained with the (CAGA) 12 -luc reporter (data not shown). Given that in TGF-b signaling, phosphorylation of the C-terminal serine residues (SSXS) is essential for dissoci- ation from the type I receptor and for heteromeric complex formation with Smad4 [34,49], we investigated C-terminal phosphorylation in response to TGF-b1aswellasNGF. The phosphorylation pattern of Smad3 resulting from stimulation with either TGF-b or NGF was analysed in Fig. 2. PC12 cells express low levels of endogenous TbRII. (A) C-terminal phosphorylation of Smad2 was investigated in either par- ental PC12 cells (lanes 1–3) or PC12 cells stably expressing TbRII-wt (lanes 4–6). Cells were kept in media containing 1 lgÆmL )1 doxycycline for 3 days to induce expression of TbRII-wt. Following starvation, cells were treated either with 2 n M NGF(lanes2and5)orwith200p M TGF-b1 (lanes 3 and 6) or were left untreated (lanes 1 and 4). Cell lysates were examined for Smad2 phosphorylation by immunoblotting using an antibody raised against the phosphorylated SSXS-motif of Smad2 (anti-PS2). (B) PC12 cells were plated on collagen-coated dishes and were cotransfected with pRL-TK, pSBE and the indicated expression constructs. Luciferase activity was measured after starva- tion and treatment for 24 h with control medium (white bars) or with medium containing 200 p M TGF-b1 (black bars). 924 M. Lutz et al.(Eur. J. Biochem. 271) Ó FEBS 2004 TGF-b-responsive L6 rat myoblasts and in PC12 cells, which were both stably transduced with a retroviral GFP– Smad3 construct [39]. Immunoblotting using an antibody that specifically recognizes C-terminally phosphorylated Smad3 [32,33] revealed that phosphorylation of both the heterologous GFP–Smad3 and the endogenous Smad3 protein occurs in L6 cells after treatment with TGF-b1, but not with NGF (Fig. 3B). In PC12 cells, however, we did not detect any phosphorylation at the SSXS-motif of Smad3; neither in response to NGF nor in response to TGF-b1. The low amounts of TbRII expressed in PC12 cells can account for the lack of phosphorylation upon TGF-b stimulation. This suggests that NGF-mediated Smad activation occurs independently from 5 phosphorylation at the C-terminal SSXS-motif. Involvement of Smad4 in NGF-triggered activation of the Smad signaling cascade In TGF-b1-induced signaling, R-Smads form heteromeric complexes with Smad4 following the activation by TbRI. Reporter gene assays using the p3TP(+)-luc construct were performed to determine whether NGF-mediated activation of Smad response elements is also Smad4-dependent. PC12 cells were transfected with Smad3, Smad4 or a functionally inactive Smad4 variant – Smad4(DSAD) – either alone or in the indicated combinations. Smad4(DSAD) lacks amino acids 274–321 which encode the Smad activation domain (SAD) [35,50]. Figure 4A demonstrates that ectopic expres- sion of Smad3 results in efficient transcriptional activation of Smad-dependent reporter genes, whereas neither Smad4 nor the mutant Smad4 show an effect on luciferase induction when expressed alone. Coexpression of Smad3 and Smad4, however, enhances the Smad3 effect. In Fig. 3. Mode of Smad activation by NGF. (A) Induction of specific R-Smads was investigated using the p3TP-luc(+) reporter. PC12 cells were transiently transfected with the indicated Smad constructs. Fol- lowing starvation, cells were left untreated (white bars) or were stimu- lated with 2 n M NGF (black bars). Data were normalized as described inMaterialsandmethods.Errorbarswerecalculatedfromthree independent measurements. (B) Phosphorylation of Smad3 was tested in TGF-b responsive L6 rat myoblasts (lanes 1–4) and PC12 cells (lanes 5–8) that were transduced with pMX-GFP-Smad3 (lanes 2–4 and 6–8) using retroviral transfer. Cells were starved for 4 h, followed by sti- mulation with 2 n M NGF (lanes 3 and 7) or 200 p M TGF-b1(lanes4 and 8) and cell lysates were analysed for C-terminally phosphorylated Smad3 (upper panel) or for total amounts of Smad3 (lower panel). Fig. 4. Role of Smad4 in NGF-triggered activation of the Smad path- way. (A) The role of functional Smad4 was investigated by reporter gene assays using p3TP-luc(+). PC12 cells were cotransfected with the luciferase constructs and with different combinations of Smad3 and Smad4 variants as indicated. Following starvation and treatment for 24 h with either 2 n M NGF (black bars) or control medium (white bars), cell lysates were prepared and used for luminescence measure- ment. (B) NGF-induced association of Smad3 and Smad4 was assessed by coimmunoprecipitation studies. PC12 cells were starved and treated with 2 n M NGF for the indicated periods of time. Smad3 was immunoprecipitated from cell lysates using the anti-Smad2/3 Ig and analyzed by Western blotting using anti-Smad4 Igs. Additionally, the experiment was repeated with immunoprecipitation of Smad4 followed by detection of Smad3 by Western blotting (upper panels). Total amounts of protein were verified by immunoblotting proteins of total lysates using the appropriate antibodies (lower panels). Ó FEBS 2004 Crosstalk between NGF and TGF-b pathways (Eur. J. Biochem. 271) 925 contrast, coexpression of the functionally inactive Smad4 variant – Smad4(DSAD) – largely prevents Smad3- mediated reporter gene activation. These results were also confirmed by using the (CAGA) 12 -luc reporter (data not shown). Furthermore, co-immunoprecipitation studies confirmed that NGF stimulation of PC12 cells leads to interaction between Smad3 and Smad4 (Fig. 4B). Whereas in the absence of ligand there is no heteromeric complex forma- tion, NGF treatment triggers association of the Smad proteins within 30 min, indicating that NGF directly activates Smad signaling. NGF stimulation rapidly initiates nuclear accumulation of Smad3 To assess whether Smad3 translocates to the nucleus in response to NGF treatment, nuclear extracts were investi- gated for the content of Smad3 protein and the cellular distribution of Smad3 6 was determined in whole cells. Nuclear extracts were prepared from L6 rat myoblasts that were stimulated with TGF-b1 (Fig. 5, upper panel) and from PC12 cells at several time points after NGF treatment (Fig. 5 7 , middle panel). In both cell lines, Smad3 can be detected in the nuclear fraction after 5 min of ligand stimulation and the amount of nuclear Smad3 increases with prolonged growth factor treatment, reaching a maxi- mum after 15–30 min of stimulation with TGF-b1inL6 cells and after 1 h of stimulation with NGF in PC12 cells. Referring to Smad4, there are significant levels of protein in the nucleus of both cell lines already in the absence of ligand. Cycloheximide treatment for 1 h prior to the addition of NGF indicated that Smad3 was directly stimulated by NGF for nuclear translocation, with no de novo protein synthesis required (Fig. 5, lower panel). Next, we investigated the nuclear transport of Smad3 in PC12 cells by confocal microscopy. Staining with Hoe- chst 33342 was performed to visualize the nuclei. Without NGF treatment, Smad3 is distributed throughout the whole cell (Fig. 6, first row). Stimulation with NGF for 30 min shows a strong decrease of cytoplasmic Smad3 staining and accumulation of Smad3 in the nucleus, and NGF treatment for 3 h results in a solely nuclear localization of Smad3 (Fig. 6, second and third rows, respectively). Cycloheximide treatment indicated that Smad3 was immediately stimulated by NGF for nuclear translocation; this process does not require de novo protein synthesis (Fig. 6, rows 4 and 5). This agrees with the data that we obtained by cellular fraction- ation of parental PC12 cells (Fig. 5). NGF-mediated activation of Smad reporter constructs can be efficiently abrogated by either kinase-dead TrkA receptors or by the inhibitory Smad7 protein To assess the involvement of TrkA receptors in the activation of the Smad pathway, different TrkA variants were tested in reporter gene assays using the pSBE-luc construct (Fig. 7). In PC12 cells transfected with wild-type TrkA (TrkA-wt), the basal level of luciferase activity is elevated already but the signal can be potently enhanced by stimulation with NGF. Transfection of the TrkA variant (TrkA–K538A) that carries a mutation resulting in the inactivation of the tyrosine kinase activity causes a signifi- cant reduction of responsiveness. An even stronger inhi- bitory effect can be observed after cotransfection of the wild-type TrkA receptor together with Smad7. The antago- nizing impact of Smad7 becomes additionally apparent by the strong inhibitory effect on endogenous signaling that is elicited following expression of ectopic Smad7 (Fig. 7, lanes 2 and 4). These results suggest functional TrkA receptors to be necessary for NGF-mediated activation of Smad- dependent reporter genes and demonstrate the inhibitory role of Smad7 on this NGF-mediated effect. Discussion Originally, Smad proteins were exclusively attributed to pathways activated by TGF-b family members but it becomes increasingly evident that multiple signaling cas- cades originating from other receptor systems are involved in modulating Smad signaling [14,27,30,51,52]. In the present work, we demonstrate that in PC12 cells NGF- stimulated signaling via the TrkA receptor leads to activa- tion of the Smad pathway. NGF-mediated Smad activation is independent of TGF-b ligand and occurs by a mechanism which is different from that induced by TGF-b. PC12 rat pheochromocytoma cells represent a widely used model system to investigate neuronal differentiation that is initiated following stimulation with NGF [36]. Fig. 5. 10 NGF induces nuclear accumulation of Smad3. L6 cells and PC12 cells were starved for 4 h in medium containing 0.2% (v/v) fetal bovine serum or horse serum, respectively, and nuclear fractions were pre- pared at various time points after exposure to either 200 p M TGF-b1 or 2 n M NGF as indicated. Proteins contained in the nuclear fraction were subjected to SDS/PAGE, electrotransferred to nitrocellulose and immunoblotted with monoclonal antibodies to Smad2/3 or Smad4. The purity of the cytoplasmic and nuclear fractions was confirmed by immunoblotting with an anti-lamin serum. Control cells were stimulated with 2 n M NGF for 1 h following a 1 h treatment with cycloheximide (lower panel). Nuclear fractions were probed with anti- Smad2/3 and subsequently with anti-lamin. 926 M. Lutz et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Although TGF-bs do not promote survival or differenti- ation of neuronal populations on their own, they elicit a neurotrophic potential if they are applied together with other cytokines (GDNF, GDF-5) or neurotrophins (NGF, NT-3) [4,5], suggesting that the signaling cascades of TGF-bs and neurotrophins are somehow interdependent. However, ectopic expression of inhibitors of the TGF-b/ Smad pathway such as Smad7 or neutralizing TGF-b antibodies did not prevent NGF-induced neurite formation (data not shown), suggesting that the Smad pathway that Fig. 6. Smad3 nuclear translocation in PC12 cells using confocal microscopy. Cells were plated on collagen-coated dishes, starved in medium containing 0.2% (v/v) horse serum for 4 h and stimulated for with 2 n M NGF for 30 min (second row), 3 h (third row) or were left untreated (first row). Control cells were stimulated with 2 n M NGFfor30minfollowinga1htreatmentwithcycloheximide(CHX;fourthrow)ordimethyl- sulfoxide (DMSO; fifth row). Cells were fixed, nuclei were stained with Hoechst 33342 for 2 min and the cells were analysed by confocal microscopy. The projection of multiple sections is seen on the left for each panel to visualize the morphology of the cells. The middle row shows staining of Smad3 and on the right an overlay of Smad3 and Hoechst staining is seen. Ó FEBS 2004 Crosstalk between NGF and TGF-b pathways (Eur. J. Biochem. 271) 927 can be activated by NGF is mainly important for other cellular responses. PC12 cells show transcriptional activation of TGF- b-responsive reporter genes upon NGF but not TGF-b1 stimulation (Fig. 1). Low amounts of TbRII expressed in these cells can account for TbRII being the limiting factor for proper TGF-b1 signaling, which is in accordance with results showing that ectopic expression of TbRII restores TGF-b responsiveness (Fig. 2). As an earlier report shows upregulation of TGF-b by NGF [46], we investigated whether NGF-triggered Smad activation is caused by autocrine action of TGF-b. Considering the limiting amount of TbRII discussed above, PC12 cells are equally resistant to signals evoked by either exogenous or autocrine TGF-b. Furthermore, even if all TGF-b isoforms are neutralized by the addition of antibodies or the soluble extracellular domain of TbRII-B (TbRII-B-ECD), NGF is still capable of activating Smad-dependent reporter genes (Fig. 1B,D). Concerning the amounts of secreted TGF-b,we found that besides latent (i.e. biologically inactive) TGF-b, only marginal amounts of active TGF-b can be detected in the supernatant of NGF-treated PC12 cells. TGF-b is synthesized as a precursor proprotein that is cleaved during secretion. However, the mature TGF-b remains associated with its propeptide thereby forming a latent complex until activation [53]. Because TGF-b activation takes place in the extracellular compartment, intracellular signaling events initiated by autocrine TGF-b can be excluded. Taken together, the effects of NGF on the Smad pathway are independent of TGF-b ligand. To characterize the point of convergence and the mode of Smad activation, C-terminal phosphorylation, heteromeric complex formation with Smad4 and nuclear translocation of R-Smads was investigated. Comparison of signaling through different R-Smad proteins revealed that Smad3 mediates the most potent activation (Fig. 3A). As Smad2 contains an additional exon in the MH1 domain that is not present in Smad3, it lacks the capacity to bind directly to DNA [16,17], which might explain the different behavior of the two TGF-b-activated Smads in response to NGF. Although treatment of PC12 cells with NGF results in the activation of Smad-dependent reporter genes, the preceeding signaling events are not identical to those that are known from TGF-b signaling as NGF does not induce phosphorylation of the C-terminal SSXS-motif (Fig. 3B). Recent publications describe alternative mechanisms of Smad activation which are likewise independent of C-terminal phosphorylation: c-Jun N-terminal kinase was showntoberapidlyactivatedbyTGF-b stimulation in a Smad-independent manner and to cause initial phosphory- lation of Smad3 at sites other than the SSXS motif. This modification in turn promotes TbRI-dependent C-terminal phosphorylation of Smad3 [29]. The mitogen-activated protein kinase kinase kinase was shown to trigger phosphorylation outside the C-terminal motif, which results in enhanced transcriptional activity of Smad2 in endothelial cells [54]. These examples support our findings that activation of Smad proteins can occur independently of C-terminal phosphorylation. Besides direct phosphory- lation events, NGF potentially triggers other modifications of R-Smads resulting in Smad nuclear translocation and transcriptional activation. As the NGF-initiated processes were shown to be dependent on functional Smad4 proteins (Fig. 4A) and to lead to heteromeric complex formation between Smad3 and Smad4 (Fig. 4B), we assume that the presence of the SSXS-motif is crucial to allow interaction between R-Smads and Smad4, even if the C-terminal serines are not phosphorylated. Recent reports show that phosphory- lation of the SSXS-motif enhances heteromeric complex formation and stabilizes the assembly of the Smad homo- and hetero-oligomers. Nevertheless, Smad3 and Smad4 were shown to heterotrimerize in the absence of phos- phorylation [55,56]. Thus it remains to be elucidated whether phosphorylation of other residues or different modifications causes the same or even a distinct oligomerization pattern of Smads. Nuclear translocation of Smad3 could be confirmed by the appearance of the Smad3 protein in nuclear extracts following NGF stimulation (Fig. 5) and by investigation of the cellular distribution of Smad3 by confocal microscopy (Fig. 6). The observation, that Smad3 appears in the nuclear fraction of PC12 cells after only 5 min of NGF stimulation and reaches a maximum after 1 h hints of a direct effect of NGF on Smad proteins. This is also confirmed by cycloheximide treatment (Figs 5 and 6), demonstrating that no de novo protein synthesis is required for NGF-mediated nuclear translocation of Smad3. Whereas Smad3 is not present in the nucleus in the absence of NGF, Smad4 can be found in the nucleus regardless of ligand stimulation. This is in accordance with the findings that Smad4 continuously shuttles between the cytoplasm and the nucleus [57]. R-Smads, however, underlie cytoplas- mic retention in the absence of ligand due to their interaction with Smad anchor for receptor activation 8 [58,59] or microtubules [60]. Confocal microscopy studies additionally confirm the NGF-induced nuclear transloca- tion of Smad3 within 30 min (Fig. 6). While untreated cells reveal Smad3 staining throughout the whole cell, stimula- tion with NGF for 30–60 min provokes complete nuclear accumulation of Smad3. To define the role of the high-affinity NGF receptor, TrkA, we ectopically expressed functionally inactive NGF receptors in PC12 cells and found that the tyrosine kinase Fig. 7. Functional TrkA receptors are essential for the activation of Smad-dependent reporters by NGFPC12. Cells were transfected with pSBE-luc and the indicated DNA constructs. Total amounts of DNA were kept constant by the addition of empty vector (pcDNA3). The experiment was carried out as described in Fig. 1 and error bars are calculated from three independent measurements. 928 M. Lutz et al.(Eur. J. Biochem. 271) Ó FEBS 2004 function of the TrkA receptor is required for the activation of Smad-dependent reporter constructs by NGF (Fig. 7). Interestingly, expression of Smad7 results in an almost complete loss of transcriptional activity, even when it is coexpressed with functional TrkA receptors. This demon- strates that Smad7 functions downstream of TrkA to block Smad signaling. Different scenarios of Smad7-mediated signal abrogation have been previously described. Smad7 is capable of blocking Smad signaling at the receptor level by interaction with activated TbRI [19] or by recruiting the E3 ubiquitin ligases Smurf1 and Smurf2 to the receptors, resulting in enhanced turnover of TGF-b receptors [21,22]. Furthermore, Smad7 was shown to interfere with signal transduction by interaction with cytoplasmic proteins such as TAB1 [61] or mitogen-activated protein kinase kinase kinase [54]. These distinct antagonizing mechanisms of Smad7 open up the question whether Smad7 blocks NGF- induced Smad signaling at the receptor level or by interaction with other proteins. As dominant-negative TGF-b receptor mutants did not block NGF-induced Smad activation (data not shown), they seem to be dispensible for NGF-mediated signals, and therefore a mechanism that involves Smad7 interaction with cytoplas- micproteinsisfavored. The Alk7 type I receptor is highly similar in its intracellular domain to TbRI and the constitutively active form of Alk7 was shown also to induce Smad2/3 phos- phorylation. Studies in PC12 cells have indicated that Alk7 signaling augments differentiation response to NGF [48]. The ligand for this receptor, however, is presently unknown. In conclusion, we describe here that NGF stimulation of PC12 cells results in activation of the Smad pathway independently of TGF-b1. This activation is direct and results in nuclear translocation of Smad3 within only 30 min of NGF treatment. Binding of NGF to its high- affinity receptor TrkA induces activation of Smad3, heteromeric complex formation with Smad4, nuclear trans- location and transcriptional activation. However, unlike TGF-b1 signaling, this process does not include phosphory- lation of the C-terminal SSXS-motif of the R-Smad. Based on the diverse mechanism of Smad activation by either TGF-b1 or NGF, specific subsets of target genes might be induced. The potential of NGF to activate the Smad pathway independently of TGF-b might be of special importance in regulating the expression of genes that are essential for the development and function of neuronal cells or other NGF-sensitive cells, in particular those which are TGF-b resistant. Acknowledgements R. 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NGF mediates the activation of the Smad pathway independently of the TGF-b ligand. TGF-b resistance is due to limiting amounts of TbRII in PC12 cells The