Smad7 and protein phosphatase 1α are critical determinants in the duration of TGF-β/ALK1 signaling in endothelial cells
© Valdimarsdottir et al; licensee BioMed Central Ltd. 2006
Received: 09 January 2006
Accepted: 29 March 2006
Published: 29 March 2006
In endothelial cells (EC), transforming growth factor-β (TGF-β) can bind to and transduce signals through ALK1 and ALK5. The TGF-β/ALK5 and TGF-β/ALK1 pathways have opposite effects on EC behaviour. Besides differential receptor binding, the duration of TGF-β signaling is an important specificity determinant for signaling responses. TGF-β/ALK1-induced Smad1/5 phosphorylation in ECs occurs transiently.
The temporal activation of TGF-β-induced Smad1/5 phosphorylation in ECs was found to be affected by de novo protein synthesis, and ALK1 and Smad5 expression levels determined signal strength of TGF-β/ALK1 signaling pathway. Smad7 and protein phosphatase 1α (PP1α) mRNA expression levels were found to be specifically upregulated by TGF-β/ALK1. Ectopic expression of Smad7 or PP1α potently inhibited TGF-β/ALK1-induced Smad1/5 phosphorylation in ECs. Conversely, siRNA-mediated knockdown of Smad7 or PP1α enhanced TGF-β/ALK1-induced signaling responses. PP1α interacted with ALK1 and this association was further potentiated by Smad7. Dephosphorylation of the ALK1, immunoprecipitated from cell lysates, was attenuated by a specific PP1 inhibitor.
Our results suggest that upon its induction by the TGF-β/ALK1 pathway, Smad7 may recruit PP1α to ALK1, and thereby control TGF-β/ALK1-induced Smad1/5 phosphorylation.
Transforming growth factor-β (TGF-β) elicits its cellular effects through activation of type I and type II serine/threonine kinase receptors [1, 2]. The constitutively active type II receptor phosphorylates specific serine and threonine residues in the juxtamembrane region (so-called GS domain) of the type I receptor. Type I receptor acts downstream of type II receptor (Tβ R-II) and has been shown to determine signaling specificity within the heteromeric receptor complex. In most cell types, TGF-β signals via TGF-β type I receptor (Tβ R-I), also termed activin receptor-like kinase 5 (ALK5). In endothelial cells (ECs), however, TGF-β can signal via Tβ R-II and two different type I receptors, i.e. the broadly expressed ALK5 and the EC-restricted ALK1. Whereas ALK5 inhibits EC migration and proliferation, ALK1 stimulates both these processes . The activated type I receptor propagates the signal through phosphorylation of specific receptor-regulated Smads (R-Smads). Whereas ALK5 induces the phosphorylation of Smad2 and Smad3, ALK1 mediates the activation of Smad1 and Smad5 [4, 5]. Activated R-Smads can assemble into heteromeric complexes with common partner (Co-) Smad, i.e. Smad4 and translocate into the nucleus where they regulate the transcription of target genes [1, 2].
I-Smads (Smad6 and Smad7) are natural inhibitors of TGF-β signaling that prevent the activation of R- and Co-Smads [6–8]. They do so by interacting efficiently with the activated type I receptors preventing access and phosphorylation of R-Smads by the activated type I receptors. Smad6 has also been found to exert its inhibitory effect on signaling by competing with Smad4 for heteromeric complex formation with activated Smad1  and by recruiting the co-repressor CtBP and thereby repress transcription [10, 11]. I-Smads were found to interact with Smad ubiquitination-related factors, Smurfs, which are HECT-domain ubiquitin ligases that target the TGF-β receptors for degradation [12, 13]. The expression of I-Smads is quickly induced upon stimulation by members of the TGF-β family and upon shear stress of the endothelium [14, 15]. Thus, I-Smads may be part of negative feedback control mechanisms.
A key event in TGF-β signaling is serine phosphorylation of Tβ R-I by Tβ R-II, and of R-Smads by Tβ R-I. These phosphorylations are tightly controlled, e.g. the immunophilin FKBP-12 binds to Tβ R-I and thereby inhibits phosphorylation of Tβ R-I by Tβ R-II . C-terminal phosphorylation of Smad2 and Smad3 is strongly facilitated by Smad anchor for receptor activation (SARA).
Serine/threonine protein phosphatases (PPs) are likely involved in the dephosphorylation of these phosphorylated signaling components. PPs consist of a catalytic subunit that binds to one or two regulatory subunits that generate holoenzymes with unique localizations and specificities . One of the major PPs is PP1 consisting of a PP1 catalytic subunit (PP1c) that can form complexes with more than 50 regulatory subunits . Four mammalian isoforms of the PP1c gene have thus far been described, i.e. PP1α, PP1β and two splice variants of PP1γ. Studies in Drosophila melanogaster suggest that PP1 binds to the decapentaplegic (dpp) type I receptor with the aid of SARA and negatively regulates dpp signaling . Very recently, it was reported that the TGF-β- induced Smad7 can interact with the growth arrest and DNA damage protein 34 (GADD34) (21), which is a regulatory subunit of PP1. The Smad7- GADD34 complex was shown to recruit PP1c to Tβ R-I, and thereby dephosphorylate and inactivate it .
In the present report, we have investigated the molecular mechanisms that underlie the TGF-β-induced transient ALK1-mediated Smad1/5 phosphorylation versus sustained ALK5-mediated Smad2 phosphorylation in ECs. Analysis of the effect of various chemical inhibitors on the TGF-β/ALK1 response, suggested an important contribution of PP1 in the negative regulation of ALK1 signaling, but not ALK5 signaling in ECs. Our data suggest that Smad7, induced by ALK1 activation, recruits PP1α to ALK1 and thereby inhibits TGF-β/ALK1-induced Smad1/5 phosphorylation in ECs.
Negative regulation of TGF-β-induced Smad1/5 phosphorylation is dependent on protein synthesis in ECs
Signal strength of TGF-β/ALK1 pathway in ECs is critically dependent on ALK1 and Smad5 expression levels
We strengthened these data by making use of the BRE-luc reporter, which can be used as a transcriptional read-out for TGF-β/ALK1 signaling . TGF-β induction of the BRE-luc reporter was potentiated upon ALK1 and/or Smad5 co-transfection (Fig. 2B) and attenuated upon siRNA-mediated knockdown of ALK1 (Fig. 2C) or Smad5 (Fig. 2D). Inhibition of TGF-β-induced activation of BRE-luc reporter by siRNA-ALK1 could be rescued by cotransfection of human ALK1 or Smad5, respectively (Fig. 2C, D). Taken together, these results demonstrate that TGF-β/ALK1-induced Smad1/5 activation is critically dependent on the ALK1 and Smad5 expression level in ECs.
Smad7 is induced upon TGF-β/ALK1 signaling in ECs
Smad7 is a highly efficient negative regulator of TGF-β/ALK1 signaling in ECs
Next, we investigated whether overexpression of Smad7 affects TGF-β/ALK1 signaling. The cells were infected with adenovirus expressing either lacZ or Flag-Smad7 at the MOI of 1000 and the effect on Smad phosphorylation was analysed after subjecting the cells to different concentrations of TGF-β. Smad7 overexpression blocked both Smad1/5 and Smad2 phosphorylation (Fig. 3B, left panel). Interestingly, if cells were infected with F-Smad7 at a MOI of 400, resulting in cells with lower levels of ectopically expressed Smad7 protein, the ALK1/Smad1/5 pathway but not the ALK5/Smad2 pathway was inhibited (Fig. 3B, right panel). We also examined whether Smad7 can inhibit Smad-mediated transcriptional responses in ECs. Smad7 inhibited TGF-β/ALK1-induced BRE-luc activity in a dose dependent manner (Fig. 3C). TGF-β-induced Smad1/5 phosphorylation was also examined when endogenous Smad7 expression was specifically inhibited. This was done by stable transfection in MEEC with siRNA-Smad7 plasmid including a hygromycin cassette. TGF-β-induced Smad1/5 phosphorylation was found to be prolonged in MEECs stably transfected with siRNA-Smad7 compared to control (PGK-hygromycin transfected) cells (Fig. 3D). Consistent with this finding, upon siRNA-mediated knockdown of Smad7, TGF-β-induced BRE-luc reporter activation was significantly enhanced. siRNA-mediated knockdown of Smad7 moderately inhibited TGF-β/ALK5 signaling using (CAGA)12-luc as read-out (Fig. 3E, right panel). The latter effect is likely indirectly caused by increased TGF-β/ALK1 signaling that antagonizes ALK5 signaling . Taken together, these data indicate that Smad7 is enhanced by TGF-β/ALK1 signaling and that it is highly effective in inhibiting the same pathway.
Inhibition of proteasome and protein phosphatase activity prolongs the TGF-β-induced Smad1/5 phosphorylation in ECs
To examine the involvement of the proteasome pathway in negative regulation of TGF-β/ALK1 signaling we treated BAECs with the proteasome inhibitor MG-132, and observed that Smad1/5 phosphorylation was stronger and prolonged compared to the non-treated cells (Fig. 1A, third panel). Interestingly, when we treated BAECs with the serine/threonine phosphatase inhibitor, calyculin, Smad1/5 phosphorylation was sustained (Fig. 1B). We also treated the BAECs with the phosphatase inhibitor, orthovanadate for 30 min. TGF-β-induced Smad1/5 phosphorylation was stronger and prolonged compared to non-treated cells (Fig. 1A, fourth panel). Orthovanadate is frequently used as a tyrosine phosphatase inhibitor, but certain PPs, such as the PP1 serine/threonine phosphatase, are also potently inhibited by orthovanadate . These findings suggest that proteasome degradation and dephosphorylation by PPs play prominent roles in inhibiting the activation of TGF-β-induced Smad1/5 phosphorylation. As the involvement of proteasome pathway, but not PPs, had been intensely investigated in TGF-β signaling, we focused our subsequent studies on the involvement of PPs in TGF-β/ALK1 signaling.
PP1α, which is transcriptionally induced by ALK1 activation, negatively regulates ALK1 signaling in ECs
PP1α binds strongly to ALK1 in the presence of Smad7 and dephosphorylates the ALK1 kinase
In the present study we have investigated the molecular mechanisms that underlie the transient Smad1/5 activation by TGF-β/ALK1 signaling versus the sustained TGF-β/ALK5-induced Smad2 activation in ECs . Our results support a model in which the inhibitory Smad7 is specifically induced by the TGF-β/ALK1 pathway and participates in a negative feedback loop in ECs. A rate equation model for the TGF-β/Smad pathway in ECs showed that Smad7 feedback loop provides robustness to the system . Smad7, previously shown to be capable of interacting with type I receptors [6, 8], may recruit PP1α to ALK1. The serine phosphatase activity of PP1α mediates dephosphorylation and inactivation of ALK1.
Duration of TGF-β/Smad signaling is a critical determinant for regulating specificity of cellular biological responses . During Xenopus embryogenesis differences in the duration of Smad signaling is carefully controlled since it is important for cell fate decisions . Whereas epithelial cells with a sustained Smad response are arrested in growth by TGF-β, pancreatic tumors that demonstrate a transient TGF-β/Smad response have specifically evaded anti-proliferative effects of TGF-β, while maintaining other TGF-β responses . TGF-β/ALK1 signaling promotes the activation states of ECs. A transient versus sustained ALK1 response, as determined by Smad7 and PP1α expression levels, could be of critical importance for angiogenesis. After ECs receive a TGF-β/ALK1 signal and start to proliferate, migrate and form sprouts, this signal must be turned off, whereafter the TGF-β/ALK5 signal dominates and the maturation of vessels is induced. Interestingly, PP1 (and 2A) activity was previously shown to be needed to maintain ECs in a resting state ; inhibition of PP1c activity promoted EC migration consistent with a negative role for this phosphatase in TGF-β/ALK1-induced activation of ECs.
Here we show that in ECs Smad7 is more efficiently induced by ALK1 than by ALK5. In addition, Smad7 is more efficient in blocking signaling via ALK1 than ALK5. Previously, Smad7 was shown to be induced by, and to be a general inhibitor of, TGF-β superfamily signaling in various non-ECs [14, 28, 29]. Specific factors present in ECs are likely to be the reason why Smad7 is much more important feedback inhibitor downstream of ALK1 than ALK5 signaling in ECs, compared to TGF-β and BMP signaling in other cell types.
The proposed mechanism by which PP1 mediates the dephosphorylation of ALK1 in ECs is reminiscent to that recently reported for the recruitment of PP1 by Smad7 to ALK5 in epithelial cells . Shi and co-workers reported that Smad7 interacts with GADD34, a regulatory subunit of PP1 holoenzyme. PP1c is recruited to ALK5 via a Smad7-GADD34 complex and then dephosphorylates activated ALK5. SARA enhances the recruitment of PP1c to the Smad7-GADD34 complex by enhancing the availability of PP1c to the Smad7-GADD34 complex. Which regulatory subunit of PP1α holoenzyme cooperates with Smad7 to interact with ALK1 remains to be investigated.
Our results suggest that upon its induction by the TGF-β/ALK1 pathway, Smad7 recruits PP1α to ALK1, and thereby inhibit TGF-β/ALK1-induced Smad1/5 phosphorylation. Smad7 functions in different ways to exert its antagonistic effects. Besides the recruitment of PP1 by Smad7 to the phosphorylated type I receptor to dephosphorylate and inactivate it, Smad7 has been shown to compete with R-Smads for binding to activated type I receptors and thereby inhibit phosphorylation of R-Smads [6, 8]. Our results do not exclude the possibility that ALK1/Smad signaling is subject to this type of inhibitory regulation by Smad7. In addition, Smad7 can recruit Smurf E3-ubiquitin ligases to the activated type I receptor, resulting in receptor ubiquitination and degradation [12, 13]. Treatment of ECs with proteasome inhibitor revealed that TGF-β/ALK1 signaling is also negatively regulated by the proteasome pathway (Fig. 1); whether this occurs at receptor or Smad level remains to be elucidated. Further experiments are needed to examine the contribution of PP1 compared to other mechanisms of negative control by Smad7 in TGF-β/ALK1 signaling.
Ligands, antibodies and inhibitors
Recombinant TGF-β 3 was obtained from K. Iwata (OSI Pharmaceuticals). Phospho-Smad1/5 and phospho-Smad2 antibodies that specifically recognize phosphorylated Smad1/5 and phosphorylated Smad2 have been previously described [3, 30], Smad5 rabbit antisera , Flag antibodies (SIGMA), HA antibodies (Roche), α-actin antibodies (Chemicon), goat-PP1α (Santa Cruz) and mouse-PP1c antibodies (Santa Cruz), were used in immunoblotting. Cells were pre-treated prior to stimulation with TGF-β and during the stimulation with cyclohexamide (SIGMA), MG-132 (Calbiochem), sodium orthovanadate (SIGMA-Aldrich) or calyculin (Calbiochem). In the phosphatase inhibition assay, a human recombinant protein phosphatase Inhibitor-2 (I-2) (Calbiochem), was added.
Expression plasmids and RNAi
Constructs for TGF-β signaling components have been described previously (Goumans et al., 2002). Rabbit peGFP-C1 PP1α was used in transient transfections. RNAi -Smad7 was made by cloning 5'-gatccccGACTCGCGTGGGGAGGCTCttcaagagaGAGCCTCCCCACGCGAGTCtttttggaaa-'3 and complementary oligonucleotides derived from mouse Smad7 in pSuper, RNAi-ALK1 was made by cloning 5' gatccccCACGGCTCCCTCTATGACTttcaagagaAGTCATAGAGGGAGCCGTGtttttggaaa-'3 and complementary oligonucleotides derived from mouse ALK1 in pSuper and RNAi-Smad5 was made by cloning 5'-gatccccGGTGTTCATCTATACTACGttcaagagaCCGTAGTATAGATGAACACCtttttggaaa-'3 and complementary oligonucleotides derived from mouse Smad5 in pSuper . To silence endogenous PP1α expression, double-stranded 21-nt RNAs directed against Smad7 were chemically synthesized and purified (Qiagen). The siRNA-PP1α sequence was 5'-AAGACGUUCACUGACUGCUUC-'3.
Bovine aortic ECs (BAEC) were routinely cultured in low-glucose DMEM (Gibco BRL) supplemented with 10% calf serum, L-glutamine and antibiotics. The cells were grown in a 10% CO2-containing atmosphere at 37°C. Mouse embryonic ECs (MEECs) were routinely cultured in DMEM supplemented with 10% fetal calf serum (FCS), non-essential amino acids, L-glutamine and penicillin/streptomycin on 0.1% gelatin-coated dishes. COS-7 and 293T cells were cultured in DMEM supplemented with 10% FCS, L-glutamine and penicillin/streptomycin. MEECs, COS-7 and 293T cells were grown in 5% CO2-containing atmosphere at 37°C.
Transfections and transcriptional reporter assays
MEECs were transiently and stably transfected using lipofectamine™ reagent (Invitrogen) according to the manufacturer's protocol. COS-7 and 293T cells were transfected by conventional calcium phosphate co-precipitation method. In case of siRNA, the transfection was performed using oligofectamine™ reagent (Invitrogen) according to manufacturer's instructions. Reporter assays were performed as previously described . MEECs were transfected with 0.5 μg BRE-luc  and 0.5 μg (CAGA)12-luc , in the absence or presence of an expression plasmid. In case of BRE-luc reporter, cells were stimulated with TGF-β for 6–8 h whereas (CAGA)12-luc transfected cells were stimulated for 16 h.
Adenoviral infection of ECs
ECs were infected with adenoviruses expressing LacZ, wtALK1, caALK1, caALK5, Id1 and Smad7 at a multiplication of infection (MOI) of 500 unless else is indicated. After 16 h the cells were washed and allowed to recover for 8 h prior to starvation overnight before the indicated assays.
Western blot analysis and immunoprecipitation
ECs were grown to 90% confluence. Cells were rinsed with PBS and grown in 0.5% FBS containing medium. After 16 h, cells were stimulated for 1 h with 10 ng/ml of TGF-β 3, put on ice, rinsed with PBS and lysed in lysis buffer (125 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM PMSF, 1.5% aprotinin and 1% Triton X-100). Cell lysates were separated by SDS-PAGE using 8% polyacrylamide gels, followed by wet-transfer of the proteins to Hybond-C extra nitrocellulose membranes (Amersham). Non-specific binding of proteins to the membrane was blocked in TBS-T (0.01 M Tris-HCl, pH 7.4, 0.15 M NaCl, 0.1% Tween-20) containing 3% dry milk. Primary antibodies were diluted 1000-fold in TBS-T and secondary horseradish peroxidase-conjugated goat anti-rabbit or mouse IgG antibody (Amersham) was used at a 10,000-fold dilution in TBS-T. Detection was performed by ECL. To detect heteromeric complex formation between Smad7 and PP1α, cell lysates from transfected COS-7 and MEECs were subjected to immunoprecipitation followed by Western blotting, as previously described .
RNA isolation and reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was isolated from MEECs using RNeasy columns (Qiagen) according to manufacturer's instructions. RT-PCR reactions were performed as described by Goumans et al. (3). The PCR reactions were performed using a PTC-200 Peltier thermal cycler (MJ Research). The DNA sequences of the PCR primers that were used are available on request.
Cells were infected with the indicated viruses. After lysis, immunoprecipitation was performed using anti-HA antibodies, followed by in vitro kinase assay as described by Itoh et al.  using kinase buffer (40 mM Hepes, pH 7.4, 40 mM MgCl2, 2 mM MnCl2, 2 mM DTT) including 2.5 μCi of [32P]γ ATP, and an incubation time of 30 min. Samples were washed with lysis buffer before they were subjected to phosphatase buffer (50 mM Tris, [pH 7.5], 1 mM EDTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated with or without Inhibitor-2 (Calbiochem) (10 ng/ml) at 37°C for 1 h. Protein samples were separated by SDS-PAGE using 8% polyacrylamide gels, followed by fixation and detection on a phosphoImager.
This study was supported by grants from the Dutch Cancer Society, EC QLG1-CT-2001-01032 and EC Angiotargeting Project 504743 and Netherlands Organization for Scientific Research (902-16-295) to P.t.D. We are grateful to Dr. A. Haimovitz-Friedman for BAECs, Dr. Iwata for TGF-β 3, Dr. A.Lux for adenoviral construct of wtALK1, Dr. K. Miyazono for other adenoviral constructs, Dr. M. Bollen for peGFP-PP1α constructs. We thank Midory Thorikay for expert technical assistance and Franck Lebrin for helpful discussion about the manuscript.
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