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Genes to Cells (2009) 14, 469-482. doi:10.1111/j.1365-2443.2009.01283.x
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Receptor expression modulates the specificity of transforming growth factor-β signaling pathways

 Masaru Murakami1, Hiroyuki Kawachi2, Kenji Ogawa3, Yoshii Nishino4 and Masayuki Funaba2,5

1 Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara 229-8501, Japan
2 Laboratory of Nutritional Science, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japan
3 Laboratory of Cellular Biochemistry, RIKEN, Wako, Saitama 351-0198, Japan
4 Laboratory of Veterinary Immunology, Azabu University School of Veterinary Medicine, Sagamihara 229-8501, Japan
5 Laboratory of Nutrition, Azabu University School of Veterinary Medicine, Sagamihara 229-8501, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
In current models of transforming growth factor-β (TGF-β) family signaling, type II receptors activate specific activin receptor-like kinase (ALK) type I receptors. These serine/threonine kinases activate ligand-dependent receptor regulated (R)-Smad by phosphorylating carboxy-terminal serines. We found that the receptor expression levels affected the phosphorylation and activation of the two R-Smad subclasses, activin/TGF-β-specific (AR-Smad) and bone morphogenetic protein (BMP)-specific (BR-Smad). Co-expressing constitutively active type I and type II receptors in COS7 cells resulted in the phosphorylation of both R-Smad subclasses in a ligand-independent manner. This was verified using in vitro kinase assays. In untransfected B16 melanoma cells, TGF-β1 and BMP-2 induced phosphorylation of both R-Smad subclasses, and TGF-β1 up-regulated the inhibitor of differentiation (Id) gene, which is usually regulated by BMP. By contrast, BMP-2 up-regulated plasminogen activator inhibitor-1 (PAI-1), which is an AR-Smad-regulated gene. Except for ALK4 and ALK6, levels of type I and type II receptor mRNAs were higher in B16 cells than in HeLa and HepG2 cells, in which TGF-β1 and BMP-2 induced phosphorylation of only the expected R-Smad. These results help to explain the diverse effects of this ligand family.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Transforming growth factor-β (TGF-β) family members have diverse physiological roles, including the control of cell division, early embryonic patterning, differentiation, and cell determination (Chang et al. 2002; Bilezikjian et al. 2006). Compared with these diverse functions, their signal transduction pathways are relatively simple. TGF-β ligands transmit signals through specific heteromeric complexes with type I receptors, called activin receptor-like kinases (ALKs), and type II receptors. TGF-β, which is the prototype ligand, signals via the TβRII type II receptor and uses the ALK5 type I receptor. Activin, which is another family member, associates with ActRIIA and ActRIIB type II receptors, and the ALK4 type I receptor. Signals elicited by bone morphogenetic protein (BMP), which is also a member of the TGF-β family, are mediated by BMPRII, ActRIIA and ActRIIB type II receptors, and ALK2/3/6 type I receptors. Most ligands bind with high affinity to the type II or type I receptors alone; however, some, including TGF-β2 and BMP-7, bind efficiently only to heteromeric receptor complexes (Attisano & Wrana 2002; Miyazawa et al. 2002; Feng & Derynck 2005; Massagué et al. 2005).

Both type I and type II receptors have intracellular serine/threonine kinase domains. According to current models, constitutively active (c.) type II receptor kinases transphosphorylate and activate type I receptors, which then transmit signals downstream by phosphorylating carboxy (C)-terminal serines in their physiological substrates, receptor-regulated (R)-Smads. There are two R-Smad subclasses: activin/TGF-β pathway-specific R-Smads (AR-Smads; Smad2 and Smad3) and BMP pathway-specific R-Smads (BR-Smads; Smad1, Smad5 and Smad8). Once activated by phosphorylation, R-Smads form complexes with a common partner (Co)-Smad, Smad4, and accumulate in the nucleus where they interact with transcriptional regulators for target genes (Attisano & Wrana 2002; Miyazawa et al. 2002; Feng & Derynck 2005; Massagué et al. 2005). However, it is not clear whether this simple signaling model can adequately explain the diversity of effects of the TGF-β family in different cell types, especially, when using this limited combination of ligands, type I/type II receptors and R-Smads.

Several studies have shown diverse associations between receptors and subsequent R-Smad activation, implying flexibility in the signaling pathways used by the TGF-β family. For example, the cytoplasmic domain of TβRII formed complexes with both ALK2 and ALK5 type I receptors, and transphosphorylation occurred in the absence of ligand (Chen et al. 1995; Chen & Weinberg 1995; Feng & Derynck 1996). ActRIIA also associated with both ALK2 and ALK4 in a ligand-independent manner (Willis et al. 1996). In addition, when either TβRII or ActRIIA was over-expressed with individual type I receptors in the presence of their relevant ligand, the type II receptors formed complexes with ALK1-6 (ten Dijke et al. 1994). These results suggest that increased expression of TGF-β family receptors leads to complex formation and subsequent receptor activation. Furthermore, TGF-β treatment elicited the phosphorylation of both AR-Smads and BR-Smads via ALK5 and ALK1, respectively, in endothelial cells (Goumans et al. 2002, 2003). Here we show the following: first, co-expressing type I and type II receptors induced the phosphorylation of both AR-Smad and BR-Smad; second, this phosphorylation was confirmed using in vitro kinase assays; third, AR-Smads and BR-Smads were phosphorylated and activated in untransfected B16 melanoma cells treated with BMP-2 and TGF-β1, respectively; and, fourth, receptor expression was relatively high in B16 cells. Our data show that the Smad-dependent signaling by the TGF-β family can be quantitatively and qualitatively modulated by the expression level of receptors, which may explain the diverse effects associated with these ligands partly.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Type I and type II receptor complexes phosphorylate and activate both R-Smad subclasses

Previous studies showed that over-expressing type I and type II receptors resulted in complex formation and transphosphorylation, suggesting receptor activation could occur in a ligand-independent manner (Chen et al. 1995; Chen & Weinberg 1995; Willis et al. 1996). To explore which R-Smad was phosphorylated when type I and type II receptors were over-expressed, c. forms of both receptors were expressed separately or together in COS7 cells that were co-expressing either Smad1 (as a representative BR-Smad) or Smad2 (as a representative AR-Smad). Phosphorylation of isolated Smad proteins was analyzed by Western blotting. The anti-phospho-Smad2 antibody specifically recognized the phosphorylated protein, whereas the anti-phospho-Smad1/5/8 antibody predominantly recognized the phosphorylated protein but also cross-reacted with the unphosphorylated form (Funaba & Murakami 2008).

Consistent with the current signaling model, when caALK1/2/3/6 (ALK1/2/3/6(QD)) alone was expressed, Smad1 was significantly phosphorylated (Fig. 1A). By contrast, expression of caALK4/5 (ALK4(TE) and ALK5(TD)) alone induced phosphorylation of Smad2 (Fig. 1B). Expressing type II receptors alone did not result in significant phosphorylation of Smad1 or Smad2, although TβRII expression induced weak phosphorylation of Smad2. Unexpectedly, however, over-expressing type II receptors with the caALK type I receptor together in COS7 cells induced phosphorylation of both Smad1 and Smad2. Similar results were seen with different type I and type II receptor combinations. This result was at variance with the current model of TGF-β signaling in which type I receptors were considered to determine signal specificity and which R-Smad is phosphorylated (Attisano & Wrana 2002; Miyazawa et al. 2002; Feng & Derynck 2005; Massagué et al. 2005).


Figure 1
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  		Figure 1  Effect of over-expressing type I and type II TGF-β family receptors on the phosphorylation of both R-Smad subclasses. Western blot analysis of anti-Myc antibody immunoprecipitates from COS7 cells transfected with 6Myc-Smad1 (A) or 6Myc-Smad2 (B) and the indicated caALK and type II receptor. The membranes were probed with anti-phospho-Smad1/5/8 antibody (A) or anti-phospho-Smad2 antibody (B) and were re-probed with anti-Myc antibody to check the total Smad expression. Receptor expression was monitored in Western blots of total cell lysates.

 
To check that the unconventional phosphorylation of R-Smads was directly due to the expected receptors, in vitro kinase assays were performed. Recombinant Smad proteins, purified to near homogeneity (Fig. 2A), were incubated with immunoprecipitated TGF-β family receptors expressed in COS7 cells in the presence of ATP, and phosphorylation of R-Smad was examined by Western blot analyses. When a kinase-defective mutant of either receptor was used, the immunoprecipitates did not phosphorylate Smad1 (Fig. 2B), suggesting that the kinase activities of both receptors were required for the unconventional R-Smad class to be phosphorylated. Similar results were obtained for Smad2 phosphorylation. Smad2 was phosphorylated by immunoprecipitates of ALK6(QD), but not ALK6(KR), with ActRIIB(wt) but not ActRIIB(KR) (Fig. 2C). The in vitro kinase assays showed that AR-Smad and BR-Smad could be substrates for receptor kinases in activated BMP receptor complexes or activated TGF-β receptor complexes, respectively.


Figure 2
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  		Figure 2  Direct phosphorylation of R-Smad by receptor complexes. (A) Purified Smad1 and Smad2 proteins, expressed in E. coli as GST-fused proteins, were cleaved from GST, purified and separated by SDS-PAGE. (B and C) Western blot analysis of purified Smad1 (B) and Smad2 (C) phosphorylated via in vitro kinase assays by the indicated HA-tagged caALK and type II receptor, which were expressed in COS7 cells and immunoprecipitated with anti-HA antibody (6(QD)*: ALK6(QD) and ActRIIB(wt), 5(TD)*: ALK5(TD) and TβRII(wt)). Western blots were probed with anti-phospho-Smad1/5/8 antibody (B) and anti-phospho-Smad2 antibody (C). The membranes were re-probed with anti-Smad1 antibody (B) and anti-Smad2 antibody (C) to monitor Smad protein loading.

 
TGF-β1 and BMP-2 induce BR-Smad and AR-Smad phosphorylation, respectively, in B16 cells

We next examined R-Smad phosphorylation in cell lines that had not been transfected with exogenous DNA. In the six cell lines examined, phospho-Smad1/5/8 was increased in response to BMP-2 treatment for 1 h, whereas Smad2 phosphorylation was induced by TGF-β1 treatment, consistent with the current model. Activin A and TGF-β1 had similar effects on Smad2 phosphorylation, except in MC3T3-E1 osteoblastic cells and J774-1 macrophage-like cells where activin A was ineffective (Fig. 3A). TGF-β1 also induced Smad1/5/8 phosphorylation in B16 melanoma cells, MC3T3-E1 cells, RAW264 macrophage-like cells, and J774-1 cells but not HeLa cervical cancer cells and HepG2 hepatoma cells. Previously, TGF-β has been reported to induce phosphorylation of both Smad1/5 and Smad2, but only in endothelial cells (Goumans et al. 2002). Our results extend the observation of BR-Smad phosphorylation in response to TGF-β beyond endothelial cells. In B16 cells, significant Smad2 and Smad3 phosphorylation was also seen in response to BMP-2 treatment (Fig. 3A and Supporting Fig. S1). As both patterns of unconventional R-Smad phosphorylation were seen in B16 cells, we used this cell line to pursue the mechanism and physiological relevance of R-Smad phosphorylation.


Figure 3
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  		Figure 3  TGF-β1-induced phosphorylation of BR-Smad and BMP-2-induced phosphorylation of AR-Smad in B16 cells. (A) R-Smad phosphorylation in cell lines treated with the TGF-β family ligand (200 pM TGF-β1, 4 nM activin A and 4 nM BMP-2) for 1 h (B) Changes in R-Smad phosphorylation in B16 cells with time. B16 cells were cultured with or without 200 pM TGF-β1, 4 nM activin A, or 4 nM BMP-2 for the indicated time. (C) Western blots showing the effects of increasing concentrations of TGF-β ligands on Smad phosphorylation in B16 cells. These cells were cultured with TGF-β1 (0, 0.32, 1.6, 8, 40, or 200 pM), activin A (0, 6.4, 32, 160, or 800 pM, or 4 nM) and BMP-2 (0, 6.4, 32, 160, or 800 pM, or 4 nM) for 1 h (D) Effect of SB431542 on Smad phosphorylation in B16 cells. (E) Effect of SB202190 on Smad phosphorylation in B16 cells. These cells were pre-treated with the indicated concentrations of inhibitors for 10 min before the addition of 200 pM TGF-β1, 4 nM activin A, or 4 nM BMP-2 for 1 h. Smad1/5/8 and Smad2 phosphorylation was analyzed on Western blots of cell lysates.

 
Analyzing the time-course of R-Smad phosphorylation showed a relatively rapid (approximately 30 min) and persistent (12 h) phosphorylation of Smad1/5/8, after BMP-2 treatment, and Smad2, after TGF-β1/activin A treatment (Fig. 3B). By contrast, the TGF-β1-induced phosphorylation of Smad1/5/8 was transient (30 min to 2 h), in agreement with previous observations in endothelial cells (Goumans et al. 2002). BMP-2-induced phosphorylation of Smad2 was rapid and long-lasting, being detectable within 5 min and for at least 24 h after the addition of BMP-2.

Dose-response studies showed increased phosphorylation of Smad1/5/8 and Smad2 with increasing concentrations of BMP-2 and TGF-β1/activin A, respectively. In B16 cells, significant Smad1/5/8 phosphorylation occurred with ≥ 32 pM BMP-2, and significant Smad2 phosphorylation occurred with ≥ 8 pM TGF-β1 and ≥ 160 pM activin A (Fig. 3C). Smad1/5/8 phosphorylation in response to TGF-β1 and Smad2 phosphorylation in response to BMP-2 were only detected in B16 cells at higher ligand concentrations, at ≥ 40 pM TGF-β1 and ≥ 800 pM BMP-2. This differed from the dose response of Smad1/5 phosphorylation in response to TGF-β1 seen in endothelial cells, which showed a bell-shaped curve with maximal phosphorylation induced by 10 pM TGF-β1 (Goumans et al. 2002).

The role of ALK4/5/7 in the unusual phosphorylation of R-Smads in B16 cells was investigated using the serine kinase inhibitor SB431542 (Callahan et al. 2002; Inman et al. 2002) (Fig. 3D). SB431542 also inhibits p38 MAP kinase at the higher concentrations (Callahan et al. 2002; Inman et al. 2002). Thus, it was used within the concentration (approximately 10 µM) without affecting p38 MAP kinase activity in NIH-3T3 cells (Inman et al. 2002). As expected, TGF-β1/activin A-induced Smad2 phosphorylation was lower in B16 cells pre-treated with SB431542, although BMP-2-induced Smad1/5/8 phosphorylation was unaffected. As B16 cells did not significantly express ALK7 (data not shown), this suggested that Smad2 phosphorylation was mediated by receptor complexes containing ALK4/5. Pre-treatment with SB431542 also blocked TGF-β1-induced Smad1/5/8 phosphorylation but not BMP-2-induced Smad2 phosphorylation.

SB202190 has been reported to inhibit the BMP pathway (Nishihara et al. 2003; Chen et al. 2006), although it efficiently inhibited p38 MAP kinase (Manthey et al. 1998). In contrast to previous studies, pre-treatment with SB202190 did not reduce BMP-2-induced Smad1/5/8 phosphorylation, although it partially reduced the phosphorylation at the higher dose (20 µM) (Fig. 3E). The reason of the discrepant results is unknown; it is possible that role of SB202190 in blocking of the BMP pathway is cell type dependent. Unexpectedly, it inhibited TGF-β1-induced Smad1/5/8 phosphorylation and BMP-2-induced Smad2 phosphorylation. In addition, Smad2 phosphorylation was reduced by pre-treatment with SB202190 after treatment with activin A but not TGF-β1.

Phosphorylation of BR-Smad by TGF-β1 treatment up-regulates Id expression, and BMP-2-induced AR-Smad phosphorylation up-regulates plasminogen activator inhibitor-1 (PAI-1) expression in B16 cells

BMP signals efficiently activate expression of the inhibitor of differentiation (Id) gene (Miyazono & Miyazawa 2002; Yokota & Mori 2002), although TGF-β treatment up-regulated Id genes in epithelial cells (Kowanetz et al. 2004). To explore the functional consequences of TGF-β1-induced BR-Smad phosphorylation in B16 cells, we evaluated the transcriptional activation and endogenous expression of Id. A previous study identified an element responsible for phosphorylated Smad1-induced transcription between nucleotides –1232 and –1069 in the Id1 promoter region (Korchynskyi & ten Dijke 2002). Consistent with this, BMP-2 treatment increased transcription of an Id1 reporter gene containing nucleotides –1231 to +88, but not of a reporter gene containing nucleotides –1070 to +88 (Fig. 4A). TGF-β1 treatment also slightly but significantly stimulated the transcription of Id1(–1231)-luc but not Id1(–1070)-luc. The Id1 mRNA level, which was measured by quantitative RT-PCR (qRT-PCR) analyses, was increased by treatment with TGF-β1 or BMP-2 (Fig. 4D,E), as were Id2 and Id3 mRNA levels (Supporting Figs S2 and S3). The specificity of qRT-PCR was verified by agarose electrophoresis and subsequent staining with ethidium bromide (Supporting Fig. S4), and by a single peak of dissociation (melting) curve of qRT-PCR products (data not shown). Pre-treatment with SB431542 and SB202190 blocked the up-regulation of Id genes induced by TGF-β1 (Fig. 4D,E, Supporting Figs S2 and S3). By contrast, BMP-2-induced up-regulation of Ids was unaffected by pre-treatment with SB431542, whereas SB202190 reduced, but did not completely inhibit, the induction of Id2 and Id3. The effects of SB431542 and SB202190 on TGF-β1- and BMP-2-induced Id gene expression and Smad1/5/8 phosphorylation were similar (Fig. 3D,E), suggesting that TGF-β1-induced BR-Smad phosphorylation is responsible for the transcriptional regulation of Id genes in B16 cells.


Figure 4
Figure 4
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  		Figure 4  Role of TGF-β-induced phosphorylation of BR-Smad and BMP-induced phosphorylation of AR-Smad in B16 cells. Transcriptional activation of Id1-luc (A), AR3-luc (B), and CAGA-luc (C) by TGF-β family members in B16 cells. These cells were transfected with the indicated expression plasmid and β-galactosidase, and cultured with 200 pM TGF-β1, 4 nM activin A, or 4 nM BMP-2 for 16 h. Data shown are the mean ± SD from a representative experiment (n = 3). (D–G) Effects of kinase inhibitors on Id1 and PAI-1 induction by TGF-β family members in B16 cells. Cells were pre-treated with the indicated concentrations of SB431542 (D and F) or SB202190 (E and G) for 10 min before addition of 200 pM TGF-β1, 4 nM activin A, or 4 nM BMP-2 for 1 h. Transcription levels of Id1 (D and E) and PAI-1 (F and G) were measured by quantitative real-time RT-PCR, and expressed as ratios to G3PDH, with the level in B16 cells without inhibitor or ligand set to 1. Data shown are the mean ± SD (n = 2). Number on the bar indicates a ratio to expression in B16 cells treated with the inhibitor but not with the ligand.

 
We next examined the activity of phosphorylated AR-Smad induced by BMP-2 in B16 cells. Reporter assays showed that not only TGF-β1 or activin A, but also BMP-2, stimulated AR3-luc transcription (Fig. 4B). Similar results were also obtained, when CAGA-luc was used as a reporter gene (Fig. 4C). PAI-1 gene expression is up-regulated by phosphorylated AR-Smad (Hua et al. 1999; Piek et al. 2001). Consistent with our previous study in B16 cells (Murakami et al. 2006), not only TGF-β1 or activin A, but also BMP-2, induced PAI-1 expression (Fig. 4F,G). Pre-treatment with SB431542 blocked TGF-β1-induced and activin A-induced PAI-1 expression, but BMP-2-induced PAI-1 up-regulation was SB431542-insensitive (Fig. 4F). By contrast, pre-treatment with SB202190 did not completely inhibit TGF-β1-induced PAI-1 expression, but did inhibit activin A-induced and BMP-2-induced PAI-1 expression in a dose-dependent manner (Fig. 4G). The effects of these inhibitors on transcription were consistent with those on Smad2 phosphorylation (Fig. 3D,E). Similar regulation of gene expression was also seen in other TGF-β-regulated genes including JunB, TGIF, SnoN and Myc, although the responses to the TGF-β family were much lower (Supporting Fig. S5).

ALK5 is required for TGF-β1-induced BR-Smad phosphorylation, and ALK3–6 are involved in BMP-2-induced AR-Smad phosphorylation in B16 cells

We further explored the involvement of type I receptors in the unexpected phosphorylation of R-Smad in response to TGF-β1 or BMP-2 in B16 cells using double-stranded interfering RNA (dsRNAi) specific for particular type I receptors (Supporting Fig. S6). Transfecting dsRNAi for ALKs effectively blocked the known responses to ligands, with ALK4-specific and ALK5-specific dsRNAis efficiently blocking activin A-induced and TGF-β1-induced Smad2 phosphorylation, respectively (Fig. 5A). In addition, BMP-induced Smad1/5/8 phosphorylation was greatly reduced after transfection with ALK3-specific dsRNAi. In addition, BMP-2-induced Id gene expression was effectively blocked by transfection with ALK3 dsRNAi (Fig. 5B, Supporting Fig. S7), and activin A-induced and TGF-β1-induced PAI-1 expression, was blocked by transfection with ALK4 and ALK5 dsRNAi, respectively (Fig. 5C).


Figure 5
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  		Figure 5  Identification of the receptors responsible for phosphorylation and activation of unconventional R-Smads in response to TGF-β family members in B16 cells. (A) Western blots of B16 cell lysates showing Smad1/5/8 and Smad2 phosphorylation, in cells depleted of the indicated type I receptor by transfection with the relevant dsRNAi for 48 h, before treatment with ligand for 1 h (B and C) Effects of depleting type I receptors on Id1 (B) and PAI-1 (C) induction by the TGF-β family in B16 cells. These cells were transfected with the indicated dsRNAi for 48 h, before the addition of the TGF-β ligand for 1 h. Gene transcript levels were measured by quantitative real-time RT-PCR, and expressed as ratios to G3PDH. Number on the bar indicates a ratio to expression in B16 cells treated with the dsRNAi but not with the ligand. (D) Cell growth in response to the TGF-β family in B16 cells transfected with the indicated dsRNAi and stimulated with TGF-β ligands for 3 days before measuring viable cell numbers using MTS assays. In all experiments, the cells were treated with 200 pM TGF-β1, 4 nM activin A, or 4 nM BMP-2.

 
ALK5 knockdown effectively blocked TGF-β1-induced Smad1/5/8 phosphorylation. In endothelial cells, ALK1 has been suggested to mediate TGF-β-induced Smad1/5 phosphorylation (Goumans et al. 2002, 2003). In contrast to endothelial cells, treating B16 cells with ALK1 dsRNAi did not affect Smad1/5/8 phosphorylation induced by TGF-β1 (Fig. 5A), suggesting a different mechanism for BR-Smad phosphorylation in endothelial and melanoma cells. ALK1 dsRNAi also barely affected TGF-β1-induced Id1 expression (Fig. 5B).

In endothelial cells, TGF-β has been shown to promote cell proliferation via ALK1, but to inhibit cell growth via ALK5 (Goumans et al. 2002, 2003). Using similar experimental protocols (Goumans et al. 2003), we evaluated cell proliferation in B16 cells, by measuring viable cell numbers for 3 days after ligand treatment (Fig. 5D). TGF-β1 and activin A inhibited cell proliferation in a dose-dependent manner, whereas BMP-2 did not affect cell growth (data not shown). Transfection of dsRNAi for ALK5 and ALK4 reduced the inhibition of cell growth induced by TGF-β1 and activin A, respectively (Fig. 5D), suggesting roles for ALK4/5 as mediators of growth inhibition. ALK1 knockdown, however, did not enhance the inhibition of cell growth induced by TGF-β1, indicating that ALK1 is unlikely to promote cell growth in B16 cells.

BMP-2-induced Smad2 phosphorylation was greatly reduced by transfection with ALK3-specific dsRNAi (Fig. 5A). Compared with controls, transfection with ALK4/5/6 dsRNAi also reduced the Smad2 phosphorylation. Similarly, BMP-2-induced PAI-1 expression was also blocked by ALK3 knockdown (Fig. 5C). In addition, ALK5-specific or ALK6-specific dsRNAi reduced BMP-2-mediated PAI-1 expression. These results suggested that ALK3–6 were involved in BMP-2-induced AR-Smad phosphorylation in B16 cells, which was also verified by reporter assays using Smad2-dependent AR3-luc (data not shown).

Expression of TGF-β family receptors is higher in B16 cells than in HeLa and HepG2 cells

In view of our data on receptor expression in COS7 cells and receptor knockdown in B16 cells, we hypothesized that receptor expression levels might be related to the unconventional R-Smad phosphorylation observed. In fact, transfection of lower amount of ALK5 dsRNAi, which did not affect TGF-β-induced Smad2 phosphorylation, blocked Smad1/5/8 phosphorylation in response to TGF-β1 in a dose-dependent manner (Supporting Fig. S8). Therefore, we quantified receptor gene transcripts in the human cell lines HeLa and HepG2, and the murine cell lines B16, MC3T3-E1, RAW264, and J774-1. So that expression levels could be compared between cell lines, the number of gene transcripts was precisely measured.

Comparing receptor expression in B16 cells with HeLa and HepG2 cells, which displayed the conventional pattern of R-Smad phosphorylation showed higher receptor expression in B16 cells, with the exception of ALK4 and ALK6 in HeLa cells (Fig. 6). Higher receptor expression in B16 cells than HeLa and HepG2 cells was corroborated when numbers were expressed as a ratio to β-actin (Supporting Fig. S9). Finally, we examined R-Smad phosphorylation in response to the TGF-β family in HepG2 cells transfected with type I and type II receptors. Over-expression of both receptors induced phosphorylation of unconventional R-Smad in response to the TGF-β family, although the responses were relatively smaller (Supporting Fig. S10). It is probably due to lower transfection efficiency in HepG2 cells; endogenous R-Smad phosphorylation was examined, whereas receptors are over-expressed in the limited cells.


Figure 6
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  		Figure 6  Expression level of different TGF-β family receptors in different cell lines. Gene transcript levels of the indicated receptors were quantified in the cell lines shown, and the number of molecules was expressed as a ratio to 18S rRNA.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
This study showed that co-expressing type I and type II receptors for the TGF-β family induced phosphorylation of the C-terminal serines in AR-Smad as well as BR-Smad, thus activating both R-Smad subclasses. In vitro kinase assays demonstrated direct phosphorylation of both R-Smad subclasses by activated type I and type II receptor complexes. The phosphorylation of both R-Smad subclasses after TGF-β1 or BMP-2 treatment was detected in several cell lines; in particular, BR-Smad and AR-Smad were phosphorylated and activated in B16 melanoma cells in response to TGF-β1 and BMP-2, respectively. Both the higher levels of the TGF-β family receptor gene transcripts seen in B16 cells, and the inhibition of phosphorylation of both the conventional and unexpected R-Smad by type I receptor down-regulation using dsRNAis, suggest that the level of TGF-β family receptors expressed, which varies between cells, is involved in the extent of R-Smad phosphorylation and activation. This could partly explain the diverse effects of the TGF-β family in diverse cell types.

In endothelial cells, TGF-β induced phosphorylation of BR-Smad, which is mediated by ALK1 and dependent on ALK5 expression (Goumans et al. 2003). During the preparation of this manuscript, two studies were reported on TGF-β-induced BR-Smad phosphorylation in different cell types (Daly et al. 2008; Liu et al. in press). Daly et al. (2008) suggested that formation of ALK2/3–ALK5 complex is responsible for the unconventional R-Smad phosphorylation, which is basically consistent with the notion proposed by Goumans et al. (2003), that is, lateral signaling through mixed type I receptor complexes. By contrast, Liu et al. (2009) revealed the direct phosphorylation of BR-Smad by ALK5-TβRII receptor complex and importance of ALK5L45 loop, region responsible for R-Smad recognition. The present results in B16 cells are consistent with the results by Liu et al. (2009); in fact, neither knockdown of ALK1 nor double knockdown of ALK2/3 blocked BR-Smad phosphorylation induced by TGF-β1 (Fig. 5A and Supporting Fig. S11).

It is possible that the mechanism of the unconventional R-Smad phosphorylation is cellular context dependent. Thus, in macrophage-like cells, such as RAW264 and J774-1, expressing ALK1 highly (Fig. 6), the ALK1-ALK5 complex may have a role in the TGF-β-induced phosphorylation of BR-Smad.

The present study also indicates BMP-2-induced AR-Smad phosphorylation and activation in B16 cells. As for this unconventional R-Smad phosphorylation, our results suggest the involvement of direct phosphorylation by canonical BMP receptors as well as lateral signaling of mixed type I receptors. Considering that activated ALK6-ActRIIB directly phosphorylated Smad2 (Fig. 2C) and that knockdown of ALK3 or ALK6 blocked BMP-2-induced Smad2 phosphorylation (Fig. 5A), AR-Smad can be directly phosphorylated by BMP receptor complex. In addition, knockdown of ALK4 or ALK5 inhibited Smad2 phosphorylation induced by BMP-2 (Fig. 5A) as well as AR-Smad-mediated up-regulation of PAI-1 gene (Fig. 5C).

Melanoma cells tend to metastasize (Gupta et al. 2005). TGF-β1 and BMP-2 up-regulated both PAI-1 and Ids in B16 cells, with TGF-β1 preferentially inducing PAI-1, and BMP-2 preferentially inducing Ids. An inverse relationship between metastasis and Id2 expression has been reported in melanoma cells (Onken et al. 2006): Id2 knockdown has been shown to change the phenotype of melanomas from non-metastatic to metastatic, indicating that Id2 loss triggers progression to metastasis. The plasminogen activator system is believed to facilitate tumor metastasis by promoting invasion through tissue barriers (Stefansson et al. 2003; Chorostowska-Wynimko et al. 2004). PAI-1 is a major inhibitor of the plasminogen activator system, and its over-expression inhibited metastasis in intraocular melanomas (Ma et al. 1997). The TGF-β family ligands might thus act as negative regulators of melanoma metastasis through a ligand-dependent mechanism.

There are precedents for receptor expression levels modifying TGF-β family signaling. TGF-β reversibly induced the differentiation of normal mammary epithelial NMuMG cells from an epithelial to a fibroblastic phenotype, and the expression of dominant-negative ALK2 mutant led to unresponsiveness to TGF-β (Miettinen et al. 1994). Although ALK2 generally transmits BMP signals (Attisano & Wrana 2002; Miyazawa et al. 2002; Feng & Derynck 2005; Massagué et al. 2005), it has also been suggested to mediate TGF-β signaling in NMuMG cells, which express high levels of ALK2 (Miettinen et al. 1994).

This study clearly shows that members of the TGF-β family can phosphorylate and activate both R-Smad subclasses in cells expressing high receptor levels. The unconventional phosphorylation of R-Smad, however, cannot be solely explained by receptor expression level, e.g. significantly higher expression of ALK3/5 and TβRII in MC3T3-E1 cells than in B16 cells and the comparable expression of ALK4, ActRIIA, ActRIIB and BMPRII, irrespective of failure of Smad2 phosphorylation in response to BMP-2 treatment in MC3T3-E1 cells. Future studies are needed to explore additional factor(s) involved in the unconventional phosphorylation of R-Smad. Nevertheless, the present results clearly suggest to need modification of the current model, in which the type I receptor determines signal specificity by phosphorylating a ligand-specific R-Smad. Although type I receptors principally determine the signal specificity, the whole receptor complex may potentially be able to phosphorylate and activate both R-Smad subclasses depending on the receptor expression. Activation of both R-Smad subclasses by one ligand might be an efficient signaling mechanism by which complex physiological processes could be achieved, while saving the synthesis of additional growth/differentiation factors. Compared with the diverse biological activities mediated by the TGF-β family, the current Smad-dependent signaling model is relatively simple. The modulation of R-Smad phosphorylation and activation by the level of receptor expression could be a mechanism by which TGF-β family signaling could vary with cellular context.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Materials

The following reagents were purchased: purified TGF-β1 (Becton Dickinson); recombinant activin A and BMP-2 (R & D Systems, Minneapolis, MN); SB431542 (TOCRIS) and SB202190 (Calbiochem); rabbit polyclonal antibodies against phospho-Smad1 (Ser463/Ser465)/Smad5 (Ser463/Ser465)/Smad8 (Ser426/Ser428), phospho-Smad2 (Ser465/Ser467) and Smad2 (Cell Signaling Technology); mouse anti-Smad1 mAb (A-4; Santa Cruz Biotechnology); mouse anti-β-actin mAb (AC-15; Abcam, Cambridge, U.K.); mouse anti-Flag mAb (M2; Sigma); anti-HA (12CA5) and anti-HA (3F10) antibodies (Roche); anti-Myc (9E10) antibody (Santa Cruz Biotechnology).

Plasmids

We are grateful to the following: Dr M. Whitman for providing AR3-luc reporter plasmids; Dr L. Attisano for FoxH2; Dr P. ten Dijke for CAGA-luc; Dr K. Miyazono for caALK1-3 and ALK6 (ALK(QD)), and kinase-defective ALK1 and ALK5 (ALK(KR)); Dr L. S. Mathews for caALK4 (ALK4(TE)) and ActRIIA; Dr X.-F. Wang for caALK5 (ALK5(TD)); and Dr H. F. Lodish for TβRII. Xenopus Smad2/pGEX-2T was prepared as previously described (Funaba & Mathews 2000). Other plasmids were prepared using pcDNA3, pGL4-luc, or pGEX-6P-2.

Cells and transfections

COS7, HeLa, HepG2, MC3T3-E1 and RAW264 cells were provided by the RIKEN Cell Bank. B16 and J774-1 cells were provided by the Institute of Development, Aging and Cancer, Tohoku University. Transient transfections used PolyFect (Qiagen) for HepG2 cells, and SuperFect (Qiagen) or Lipofectamine 2000 (Invitrogen) for B16 cells.

For experiments on Smad phosphorylation and gene expression, cells were cultured in medium containing 0.2% FBS for 4 h, before the addition of ligand in medium containing 0.2% FBS. For experiments using protein kinase inhibitors, cells were cultured in medium with 0.2% FBS for 4 h, treated with vehicle (DMSO) or the indicated concentrations of inhibitors with 0.2% FBS for 20 min, and then stimulated with ligand in the presence of the kinase inhibitor. Luciferase reporter assays were performed as previously described (Funaba et al. 2003).

RNA interference

dsRNAi targeting the expression of ALKs and green fluorescent protein (GFP) controls were synthesized by Samchully Pharmaceuticals (Seoul, Korea). The coding sequences are shown in Supporting Table S1. Two sets of dsRNAi were designed to target each molecule, except for the control, and 1 : 1 mixtures of the dsRNAis were prepared. B16 cells were seeded at a density of 2.5 x 105 cells/well in six-well plates, cultured overnight, and transfected with 100 pmol dsRNAi using Lipofectamine 2000 according to the manufacturer's protocol. After 48 h, cells were cultured in medium with 0.2% FBS for 4 h and then stimulated with ligand for 1 h.

Western blot analysis and immunoprecipitation

Immunoprecipitates and Western blotting were performed as previously described (Funaba & Murakami 2008). To analyze receptor expression, total cell lysates were subjected to Western blotting using anti-HA (3F10) antibody to detect HA-tagged proteins.

In vitro kinase assay

Smad1 protein was expressed in BL21 transformed with xSmad1/pGEX-6P-2, and purified using GSH-Sepharose beads according to the manufacturer's protocol. The GST moiety was cleaved using PreScission protease (GE Healthcare). Smad2 protein was expressed and purified as previously described (Funaba & Mathews 2000). COS7 cells were transiently transfected with HA or Flag-tagged receptor expression vectors for 40 h and lysed in lysis buffer (20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethanesulfonylfluoride (PMSF), 1% aprotinin, and 1 mM Na3VO4). Receptor complexes were immunoprecipitated with anti-HA (12CA5) antibody for 4 h at 4 °C, absorbed on to protein G agarose for 1 h at 4 °C, washed twice with lysis buffer and then once with kinase buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 0.1 mM CaCl2, 0.05% Triton X-100, 1 mM DTT, 1 mM PMSF, 1% aprotinin, and 1 mM Na3VO4). The receptors were incubated for 30 min at 30 °C in kinase buffer containing 500 µM ATP and 0.45 µM purified Smad1 or Smad2 as substrate. The reaction was stopped by chilling on ice for 2 min, and the supernatant and the beads were separated by centrifuging at 600 g for 2 min. Phosphorylation of Smad in the supernatants and receptor expression in the beads were analyzed by Western blotting.

RNA isolation and qRT-PCR

RNA isolation, cDNA synthesis and qRT-PCR were previously described (Funaba et al. 2003; Murakami et al. 2006). The PCR primers used to detect PAI-1 and G3PDH were previously described (Funaba et al. 2003; Murakami et al. 2006), and the other primers are shown in Supporting Table S2.

Cell-proliferation assay

Viable cell numbers were measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega) as previously described (Funaba et al. 2006). B16 cells were seeded in triplicate in 96-well plates at 1.0 x 104 cells/100 µL and cultured overnight. Cells were transfected with dsRNAi as described above, and, after 36 h, stimulated with ligand in medium with 0.2% FBS for 3 days. MTS and an electron-coupling reagent, phenazine ethosulfate, were added 4 h before measuring the absorbance. Absorbance of cells treated without ligand stimulation was set at 100.

Comparison of the expression of TGF-β receptor genes in different cell lines

The expression of TGF-β family receptors in human and mouse cell lines was compared by qRT-PCR using DNA standard quantified precisely by an Agilent2100 bioanalyzer and a DNA 1000 Lab Chip kit (Agilent Technologies). The number of DNA molecules was calculated from the DNA mass and the molecular weight and expressed relative to 18S rRNA or β-actin.


   Acknowledgements
 
We thank Drs L. Attisano, H.F. Lodish, L.S. Mathews, K. Miyazono. P. ten Dijke, X.-F. Wang and M. Whitman for providing plasmids. We also thank Drs M. Hashimoto, A. Abe, A. Kurisaki, T. Matsui and T. Murata for discussion. This work was supported by Kakenhi from The Japan Society for the Promotion of Science, by grants for Graduate Schools from The Foundation for Japanese Private School Promotion, and by The Science Research Promotion Fund from The Promotion and Mutual Aid Corporation for Private Schools of Japan.


   Footnotes
 
Communicated by: Kohei Miyazono

* Correspondence: mfunaba{at}kais.kyoto-u.ac.jp


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 Discussion
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Accepted: 12 January 2009




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Bone morphogenetic protein receptors and signal transduction
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