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¹ Institut National de la Santé et de la Recherche Medicale, Unite 673, Hôpital St-Antoine, 75571 Paris, France
² Institut National de la Santé et de la Recherche Medicale, Unite 620, Institut Federatif de Recherche 140, Université de Rennes I, 35043 Rennes, France
³ Hybrigenics, 75014 Paris, France
⁴ Department of Biomedicine and Biotech Research and Innovation Centre, University of Copenhagen, DK-1017 Copenhagen, Denmark

Correspondence to Nathalie Théret: nathalie.theret{at}univ-rennes1.fr

Transforming growth factor-ß (TGF-ß) regulates a wide variety of biological processes through two types of Ser/Thr transmembrane receptors: the TGF-ß type I receptor and the TGF-ß type II receptor (TßRII). Upon ligand binding, TGF-ß type I receptor activated by TßRII propagates signals to Smad proteins, which mediate the activation of TGF-ß target genes. In this study, we identify ADAM12 (a disintegrin and metalloproteinase 12) as a component of the TGF-ß signaling pathway that acts through association with TßRII. We found that ADAM12 functions by a mechanism independent of its protease activity to facilitate the activation of TGF-ß signaling, including the phosphorylation of Smad2, association of Smad2 with Smad4, and transcriptional activation. Furthermore, ADAM12 induces the accumulation of TßRII in early endosomal vesicles and stabilizes the TßRII protein presumably by suppressing the association of TßRII with Smad7. These results define ADAM12 as a new partner of TßRII that facilitates its trafficking to early endosomes in which activation of the Smad pathway is initiated.

	Introduction

Top Abstract Introduction Results and discussion Materials and methods References

 
TGF-ß superfamily members are multifunctional cytokines that regulate a broad range of cellular functions, including cell proliferation, differentiation, and apoptosis (Massague et al., 2000; Derynck et al., 2001). TGF-ß signals through a heteromeric complex of two types of transmembrane Ser/Thr kinases: TGF-ß type I receptor and TGF-ß type II receptor (TßRII). TGF-ß binding to TßRII induces the recruitment and phosphorylation of TGF-ß type I receptor, which, in turn, phosphorylates the receptor-regulated Smads (R-Smads) Smad2 and Smad3. Once phosphorylated, Smad2 and Smad3 associate with the common partner Smad, Smad4, and translocate to the nucleus, where they regulate the expression of TGF-ß target genes. In contrast to R-Smads and Smad4, the inhibitory Smad, Smad7, appears to block signal transduction by preventing access of R-Smads to the TGF-ß receptor or by recruiting distinct E3 ubiquitin ligases that target the receptor–Smad7 complex for degradation (Kavsak et al., 2000; Ebisawa et al., 2001; Seo et al., 2004).

Upon TGF-ß stimulation, Smad2 is recruited to the receptor complex by an adaptor molecule called Smad anchor for receptor activation (SARA). At steady state, SARA-bound Smad2 is localized in early endosomes to which the receptor is internalized via clathrin-coated pits (Hayes et al., 2002; Di Guglielmo et al., 2003). The importance of the clathrin-mediated endocytic pathway in TGF-ß signaling is also manifested by the recent finding that cPML (cytoplasmic form of the promyelocytic leukemia protein) mediates TGF-ß signaling by facilitating recruitment of the SARA–Smad2 complex and TGF-ß receptors to early endosomes (Lin et al., 2004).

In addition to clathrin, TGF-ß receptors can also associate with caveolin (Razani et al., 2001), which leads to their internalization into caveolin1-positive vesicles with subsequent degradation through the proteasome pathway. Consistent with this notion, the caveolin1-positive vesicles were found to associate with Smad7 (Ito et al., 2004), which is known to mediate the association of the E3 ligases Smurf1 and Smurf2 to receptors, leading to their degradation.

To gain more insight into the regulation of TGF-ß signaling, we have performed yeast two-hybrid screens using TßRII as bait. ADAM12 (a disintegrin and metalloproteinase 12) was one of the TßRII interactors that exhibited specific and strong binding to TßRII. ADAM12 belongs to the ADAMs family, which are glycoproteins characterized by a multidomain structure comprised of pro-, metalloproteinase, disintegrin, cysteine-rich, transmembrane, and cytoplasmic domains (Primakoff and Myles, 2000; Seals and Courtneidge, 2003). ADAMs exhibit proteolytic, cell adhesion, and signaling properties, and perturbations of ADAM expression are associated with several human diseases, including cancers (Duffy et al., 2003). In the present study, we provide the first evidence that ADAM12 interacts with TßRII and enhances TGF-ß signaling by controlling the localization of TGF-ß receptors to early endosomes. These results reveal a new role for ADAM12 in the regulation of TGF-ß receptor trafficking.

	Results and discussion

Top Abstract Introduction Results and discussion Materials and methods References

 
Using the extracellular domain of human TßRII as bait, we performed a yeast two-hybrid screen of a human placental cDNA library. Eight different fragments of ADAM12 were found to interact with TßRII (Fig. 1 A). Two variants were previously described for ADAM12: a transmembrane glycoprotein (Yagami-Hiromasa et al., 1995) and a shorter secreted form (Gilpin et al., 1998). The common sequences shared by the overlapping fragments of the prey span the metalloproteinase and disintegrin domains common to the two variants (Fig. 1 A).

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Figure 1. TßRII interacts with ADAM12. (A) Schematic diagram of ADAM12 fragments that interact with TßRII in two-hybrid assays. (B) 293T cells were transfected with HA-TßRII in the presence or absence of Flag-ADAM12. Cell lysates were subjected to anti-Flag immunoprecipitation (IP) followed by immunoblotting (IB) with anti-HA. In this and all of the following experiments, the expression of proteins was determined by direct immunoblotting. (C) C2C12 cells transfected with GFP-ADAM12 and HA-TßRII were immunostained with anti-HA followed by TRITC-conjugated secondary IgG. The panels represent three representative fields. (D–F) Cell extracts from HSC, RD, and C2C12 cells (D) or C2C12 cells (E and F) were immunoprecipitated with anti-ADAM12 (D and F) or anti-TßRII (E) and immunoblotted with the indicated antibodies.

To examine whether the association of ADAM12 with TßRII can occur under physiological conditions, we used hepatic stellate cells (HSCs), Rhabdomyosarcoma (RD), and C2C12 cells, three cell lines that were previously described to express detectable ADAM12 (Gilpin et al., 1998; Galliano et al., 2000; Le Pabic et al., 2003). In immunoprecipitates prepared with preimmune antisera, no TßRII was coprecipitated. However, in the anti-ADAM12 immunoprecipitates, we could clearly detect TßRII coprecipitating with ADAM12 (Fig. 1 D). Formation of the endogenous ADAM12–TßRII complex was also demonstrated by anti-ADAM12 immunoblotting of anti-TßRII immunoprecipitates (Fig. 1 E). The interaction of ADAM12 with TßRII is specific because we were unable to detect an interaction between TßRII and ADAM10 or ADAM17 (Fig. 1 E), which share the structure organization with ADAM12. Similarly, we were unable to see an interaction between ADAM12 and the bone morphogenetic protein type II receptor (Fig. 1 F).

To explore the functional significance of the interaction between ADAM12 and TßRII, we investigated whether the expression of ADAM12 may influence TGF-ß–mediated transcriptional esponses. For this, we made use of the TGF-ß/Smad2-responsive reporter ARE₃-Lux (Labbe et al., 1998) and found that the expression of ADAM12 resulted in an approximately fivefold increase in TGF-ß–induced transcription (Fig. 2 A). A similar effect of ADAM12 was observed with the TGF-ß/Smad3-responsive reporter CAGA₉-Lux (approximately threefold in Fig. 2 B and sixfold in Fig. 2 C; Zawel et al., 1998).

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Figure 2. ADAM12 increases TGF-ß signaling. (A–C) HepG2 (A and B) and C2C12 (C) cells transfected with ARE3-Lux together with FAST1 (A) or CAGA9-Lux (B and C) in the presence or absence of ADAM12 were treated with or without TGF-ß. In these and all of the following reporter assays, luciferase activity was determined and normalized, and results are expressed as means ± SD (error bars) of triplicates from five independent experiments. (D) HSC, RD, or C2C12 cells were transfected with the indicated combinations of pEGFP, ADAM12 shRNA, or scrambled shRNA. 36 h later, GFP-transfected cells were sorted by FACS and exposed to TGF-ß for 16 h. The expression of endogenous PAI-1, JunB, and ADAM12 was assessed by direct immunoblotting (IB). (E and F) 293T cells were transfected with myc-Smad2, Flag-ADAM12, and HA-Smad4 as indicated. For Smad2 phosphorylation (E), cell lysates were immunoblotted with antiphospho-Smad2. For the association of Smad2 with Smad4 (F), cell lysates were immunoprecipitated (IP) with an anti-myc before immunoblotting with anti-HA. (G) C2C12 cells were transfected with the indicated combinations of pEGFP, ADAM12 shRNA, or scrambled shRNA. 36 h later, GFP-transfected cells were sorted by FACS and exposed to TGF-ß for 30 min. The phosphorylation of endogenous Smad2 was assessed by immunoblotting with antiphospho-Smad2. White lines indicate that intervening lanes have been spliced out.

To investigate the mechanism underlying the effects of ADAM12 on TGF-ß signaling, we investigated whether the expression of ADAM12 may regulate the TGF-ß–dependent phosphorylation of Smad2. We observed that exposure of cells to TGF-ß resulted in increased Smad2 phosphorylation, and this effect was further enhanced by the expression of ADAM12 (Fig. 2 E). Consistent with this, the expression of ADAM12 enhanced the ability of TGF-ß to induce assembly of the Smad2–Smad4 complex (Fig. 2 F). In addition, the depletion of endogenous ADAM12 by RNAi suppressed Smad2 phosphorylation (Fig. 2 G). Collectively, these data suggest that ADAM12 may function to enhance TGF-ß signaling by facilitating Smad2 phosphorylation and its subsequent heterodimerization with Smad4.

At least six members of the ADAM family have been demonstrated to have proteolytic activity, including ADAM12 (Loechel et al., 2000; Shi et al., 2000). In initial experiments, we found that a truncated form of ADAM12 (ADAM12-tail), which lacks the cytoplasmic domain, retains its ability to enhance TGF-ß signaling (Fig. 3 A). Therefore, we sought to investigate whether the increase in TGF-ß transcriptional activity mediated by ADAM12 may involve its catalytic activity. To approach this question, we investigated the effect of phenanthroline, a specific metalloproteinase inhibitor, on the ability of ADAM12 to enhance TGF-ß transcriptional responses. Surprisingly, exposure of cells to phenanthroline failed to suppress the effect of ADAM12 on TGF-ß–induced CAGA₉-Lux (Fig. 3 B). In another approach, we used ADAM12-E351Q, a protease inactive mutant. As shown in Fig. 3 C, the expression of ADAM12-E351Q enhanced TGF-ß–induced transcription with an activity similar to that of wild-type ADAM12. Together, these results indicate that ADAM12 enhances TGF-ß signaling through a protease-independent mechanism.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Up-regulation of TGF-ß signaling by ADAM12 does not involve its cytoplasmic domain or its protease activity. (A–C) HepG2 cells were transfected with CAGA9-Lux, ADAM12, ADAM12-tail, and ADAM12.E351Q as indicated. For A and C, cells were treated with or without TGF-ß for 16 h before lysis. Dose effects are shown in insets. For B, cells were treated with 1.10 phenanthroline in the presence or absence of TGF-ß. In all cases, luciferase activity was determined. Error bars represent SD. WT, wild type.

View larger version (55K): [in this window] [in a new window] [Download PPT slide]   Figure 4. ADAM12 facilitates clathrin-dependent TGF-ß signaling. (A) MvLu1 cells transfected with GFP-ADAM12 and HA-TßRII were immunostained with rabbit anti-HA and mouse anti-EEA1 followed by secondary staining with TRITC-conjugated anti–rabbit and Cy5-conjugated anti–mouse IgG. The subcellular localization of GFP-ADAM12 (green), TßRII (red), and EEA1 (blue) was analyzed by a confocal microscope. Colocalization of EEA1 with ADAM12, ADAM12 with TßRII, and EEA1 with TßRII appears as turquoise, yellow, and purple, respectively. The panels represent three independent representative fields. Representative areas from cells that display TßRII and ADAM12 in the EEA1 compartments are enlarged in the insets. (B and C) HepG2 cells were transfected with the indicated combinations of CAGA9-Lux, ADAM12 shRNA, ADAM12, Smad3 shRNA, or scrambled shRNA. Cells were potassium depleted before stimulation with TGF-ß, and luciferase activity was examined. (D) COS7 cells were transfected with HA-TßRII and Flag-SARA with or without ADAM12. Cells were immunostained with rabbit anti-HA and mouse anti-EEA1 followed by secondary staining with TRITC-conjugated anti–rabbit and Cy5-conjugated anti–mouse IgG. For the detection of Flag-SARA, cells were incubated with FITC-conjugated anti-Flag. Flag-SARA, TßRII, and EEA1 appear as green, red, and blue, respectively. (E) HepG2 cells transfected with CAGA9-Lux and the indicated expression vectors were treated with nystatin at 10 µM for 1 h before stimulation with TGF-ß, and luciferase activity was examined. Error bars represent SD.

To provide further evidence that ADAM12 facilitates the localization of TßRII in early endosomes, we tested the effect of nystatin, a sterol-binding antibiotic that is known to induce the redistribution of TGF-ß receptors into EEA1-positive endosomes by affecting the raft structures (Di Guglielmo et al., 2003). We reasoned that if we induce the majority of TßRII to accumulate in early endosomes by an alternative approach, such as the treatment of cells with nystatin, ADAM12 should have no further effect on TGF-ß–mediated transcription. As shown in Fig. 4 E, exposure of cells to nystatin caused a considerable increase in the TGF-ß–mediated activation of CAGA₉-Lux, and this increase was not affected by the expression of ADAM12. Under these experimental conditions, the expression of Smad3 can synergize with nystatin to enhance TGF-ß–induced transcription, arguing against the possibility that the lack of ADAM12 effect is caused by the ability of nystatin to elicit the maximum threshold level of TGF-ß signaling in this cell system. Collectively, these results suggest that ADAM12 may function as an important component in TGF-ß signaling by modulating the trafficking of the TGF-ß receptor.

The clathrin-dependent internalization into early endosomes promotes TGF-ß signaling, whereas the lipid raft–caveolar internalization pathway is required for receptor turnover. To obtain direct evidence that the accumulation of ADAM12 in early endosomes plays a role in the up-regulation of TGF-ß signaling, we examine whether the expression of ADAM12 interferes with TßRII degradation. To approach this question, we first investigated the effect of ADAM12 on TßRII ubiquitination. We observed that the coexpression of ADAM12 resulted in a substantial decrease in the ubiquitination of TßRII (Fig. 5 A). In support of this result, the expression of ADAM12 increased the steady-state levels of TßRII (Fig. 5 B). Furthermore, in pulse-chase experiments, the expression of ADAM12 resulted in a marked decrease in the turnover of TßRII (Fig. 5 C). A similar result was obtained with the cytoplasmic truncated form ADAM12-tail, which, like the wild-type counterpart, can enhance TGF-ß signaling (Fig. 5 C). As a control, we found that expression of the extracellular soluble form of ADAM12 failed to stabilize TßRII (Fig. 5 C), providing support to the theory that ADAM12 may stabilize TßRII by facilitating its intracellular redistribution from the plasma membrane to early endosomes.

View larger version (60K): [in this window] [in a new window] [Download PPT slide]   Figure 5. ADAM12 prevents TßRII degradation. (A) Cells were transfected with HA-TßRII and myc-Ub in the presence or absence of ADAM12. Cell lysates were normalized on the basis of TßRII expression, immunoprecipitated (two samples for each condition) with anti-myc, and immunoblotted with anti-HA. (B) 293T cells were transfected with HA-TßRII and increasing amounts of Flag-ADAM12. Cells were treated with TGF-ß for 18 h before lysis, and cell lysates were immunoblotted with anti-HA, anti-Flag, or antiactin. (C) 293T cells transfected with HA-TßRII and the indicated expression vectors were pulse-chased with [35S]Met/Cys, and labeled HA-TßRII was immunoprecipitated and analyzed by SDS-PAGE and autoradiography. (D) Cells were transfected with HA-TßRII, myc-Smad7, and Flag-ADAM12 as indicated. Cell lysates were normalized on the basis of TßRII expression, immunoprecipitated (two samples for each condition) with anti-myc, and immunoblotted with anti-HA. (E) C2C12 cells were transfected with the indicated combinations of pEGFP, ADAM12 shRNA, or scrambled shRNA. 36 h later, GFP-transfected cells were sorted by FACS, lysed, and normalized on the basis of TßRII expression. Then, the association of TßRII with Smad7 or Smad2 was analyzed by immunoprecipitation (IP)/immunoblotting (IB).

Concluding remarks
Overall, our data describe a new function for ADAM12 in the positive regulation of TGF-ß signaling by modulating receptor trafficking. At present, a small number of proteins that interact with TGF-ß receptors are described to regulate the trafficking and turnover of these receptors. Thus, identification of ADAM12 as a novel partner of TßRII provides new insight into the initiation of TGF-ß signaling, which takes place in early endosomes.

	Materials and methods

Top Abstract Introduction Results and discussion Materials and methods References

 
Yeast two-hybrid screening
A fragment corresponding to the extracellular domain (20–160 amino acids) of human TßRII was cloned into pBTM116. The human cDNA libraries from placenta were constructed in pGADGH. A total of 10 x 10⁶ independent colonies were screened as previously described (Colland et al., 2004). The prey fragments of the positive clones were PCR amplified and sequenced.

Cell culture and transfection
The human embryonic kidney cell line 293T, HSCs, human RD cells, mouse C2C12 cells, monkey kidney COS7 cells, and mink lung MvLu1 cells were transfected using LipofectAMINE-Plus reagent (Invitrogen) according to the manufacturer's instructions. For experiments with ADAM12 antisense, cells were incubated with 2 µM of antisense oligonucleotides to ADAM12 (CTCTCTTTTATGCCTTCT and CCCCATTCCTTTCTCC) or random control oligonucleotides (ACTACTACACTAGACTAC and GCTCTATGACTCCCAG) as previously described (Lafuste et al., 2005). For RNAi experiments, cells were transfected with 0.5 µg of expression vector encoding the indicated shRNA.

Plasmids
ARE₃-Lux, GAGA₉-Lux, FAST1, HA-Smad4, myc-Smad2, and myc-Smad7 were previously described (Dumont et al., 2003; Seo et al., 2004). The expression vector for HA-TßRII was provided by J. Wrana (Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada). Expression constructs for wild type or mutants of ADAM12 and ADAM12 fused to EGFP were prepared as previously described (Hougaard et al., 2000). The expression vector encoding ADAM12 shRNA or scrambled shRNA was constructed using the BLOCK-IT U6 RNA System (Invitrogen) according to the manufacturer's instructions. The expression vector for Flag-ADAM12 was obtained by fusing the Flag epitope to the N terminus of the ADAM12 fragment (amino acids 142–739) isolated in the yeast two-hybrid screen.

Transcriptional reporter assays
HepG2, C2C12, or 293T cells were transfected by LipofectAMINE, and, 30 h later, they were treated for 18 h with 2 ng/ml human TGF-ß1 (Sigma-Aldrich). Cell extracts were assayed for luciferase activity using the Dual Luciferase Reporter Assay System (Promega), and luciferase activities were normalized on the basis of Renilla luciferase expression from the pRL-TK control vector. For potassium depletion experiments, transfected cells were incubated in medium and water (1:1) for 5 min at 37°C followed by incubation in medium depleted or not depleted in KCl for 1 h at 37°C before stimulation with TGF-ß.

Immunoprecipitation and immunoblotting
After transfection, cells were lysed in lysis buffer (Dumont et al., 2003), and cell lysates were subjected to immunoprecipitation with the appropriate antibody for 2 h followed by adsorption to Sepharose bead–coupled protein G for 1 h. Immunoprecipitates were washed five times with lysis buffer containing 0.5% NP-40. For the association of endogenous TßRII with endogenous ADAMs, immunoprecipitates were washed three times with lysis buffer containing 0.5% NP-40 and two times with lysis buffer containing 1% NP-40. Then, samples were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. The following antibodies were used: anti-ADAM12 Rb 122 (Gilpin et al., 1998), anti-Flag M2 (Sigma-Aldrich), anti-HA and anti–myc-9E10 (Boehringer Manheim), antiphospho-Smad2 (Cell Signaling Technologies), anti-Smad2 (Zymed Laboratories), anti-ADAM10 (ProSci), anti-ADAM17 (Chemicon), and antiactin, anti-TßRII, anti–bone morphogenetic protein RII, anti-Smad7, anti–PAI-1, and anti-JunB (Santa Cruz Biotechnology, Inc.).

Immunolocalization
Cells were fixed in 3% PFA, permeabilized with 0.1% Triton X-100, and incubated for 60 min at room temperature with the appropriate primary antibody followed by the appropriate secondary antibody. The coverslips were washed, mounted in PBS containing 50% glycerol and 1 mg/ml 1,4-diazabicyclo[2.2.2]octane, and viewed on an automated microscope (DMRXA2; Leica) equipped with a camera (CoolSNAP ES N&B; Roper Scientific) and a 63x Hcx Pl Apo NA 1.32 oil objective (Leica). Z steps were submitted to deconvolution (nearest neighbor method) by using MetaMorph software (Universal Imaging Corp.).

Real-time PCR
Total RNA were extracted by the guanidinium thiocianate/cesium chloride method, and real-time quantitative PCR was performed by the fluorescent dye SYBR green methodology as previously described (Le Pabic et al., 2003). Primer pairs for target genes were as follows: PAI-1, sense (5'-GTCTTTCCGACCAAGAGCAG-3') and antisense (5'-CGATCCTGACCTTTTGCAGT-3'); ADAM12, sense (5'-GTTTGGCTTTGGAGGAAGCACAG-3') and antisense (5'-TGCAGGCAGAGGCTTCTGAGG-3'); COL1A2, sense (5'-GGTGGTGGTTATGACTTTG-3') and antisense (5'-ATACAGGTTTCGCCGGTAG-3'); and 18S, sense (5'-CGCCGCTAGAGGTGAAATTC-3') and antisense (5'-TTGGCAAATGCTTTCGCTC-3').

Online supplemental material
Fig. S1 A shows the effect of increasing amounts of ADAM12 on expression of the TGF-ß–responsive gene PAI-1. Fig. S1 B shows the TGF-ß–dependent expression of endogenous ADAM12 or COL1A2 in cells treated with ADAM12 antisense or control oligonucleotides. Fig. S2 shows the colocalization of ADAM12 with EEA1 or TßRII in Mv1Lu cells. Fig. S3 shows the association of TßRII with several transmembrane proteins as indicated by labeling with a membrane-impermeable biotinylation reagent. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200612046/DC1.

	Acknowledgments

 
We thank Dr. Stephanie Dutertre from Institut Federatif de Recherche 140 for technical assistance in microscopy and thanks the Hybrigenics staff for their contributions.

This work was supported by Institut National de la Santé et de la Recherche Médicale, Association pour la Recherche contre le Cancer (ARC), and the Danish Cancer Society. E. Dumont was a recipient of a fellowship from ARC.

Submitted: 8 December 2006

Accepted: 14 June 2007

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