ADAM17-mediated shedding of the IL6R induces cleavage of the membrane stub by γ-secretase

Biochimica et Biophysica Acta 1803 (2010) 234–245

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a m c r

ADAM17-mediated shedding of the IL6R induces cleavage of the membrane stub by γ-secretase Athena Chalaris, Jessica Gewiese, Krzysztof Paliga, Lina Fleig, Alex Schneede, Karsten Krieger, Stefan Rose-John ⁎, Jürgen Scheller Institute of Biochemistry, Christian-Albrechts-University of Kiel, Olshausenstr. 40, D-24098 Kiel, Germany

a r t i c l e

i n f o

Article history: Received 18 May 2009 Received in revised form 7 December 2009 Accepted 8 December 2009 Available online 21 December 2009 Keywords: Regulated intramembraneous cleavage ADAM17 Shedding IL6R γ-secretase

a b s t r a c t Interleukin-6 (IL6) signals are mediated by classic and trans-signaling. In classic signaling, IL6 first binds to the membrane bound Interleukin-6 Receptor (IL6R) whereas in trans-signaling, IL6 acts via a soluble form of the IL6R. Trans-signaling via the soluble IL6R (sIL6R) was linked to chronic inflammation and cancer. The release of the IL6R is mediated by the disintegrin and metalloproteinases ADAM10 and ADAM17. To analyze the fate of the C-terminal cleavage fragment after ectodomain shedding we fused the IL6R C-terminally to two Z-domains of Protein-A (2Z-tag) or to GFP. A specific C-terminal fragment of the IL6R protein could be detected after ADAM17-induced shedding. Using γ-secretase inhibitors and gene-deficient cells, we demonstrate that after ADAM17 mediated cleavage, the IL6R C-terminal fragment was cleaved by the γ-secretase at the plasma membrane. We were, however, not able to detect an IL6R intracellular domain. After γ-secretase cleavage IL6R cell surface expression was lost and γ-secretase cleavage product(s) of the IL6R were endocytosed. No GFPfluorescence of a γ-secretase-cleaved IL6R-GFP fusion protein was observed in the nucleus. We therefore hypothesize that a potential IL6R intracellular domain fragment is not involved in nuclear signaling but rapidly degraded. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Sheddases are responsible for ectodomain shedding of transmembrane proteins, resulting in the release of their extracellular domains from the cell membrane. Sheddases include members of the disintegrin and metalloprotease (ADAM) family, such as ADAM10 and ADAM17; aspartyl proteases such as the β-site APP-cleaving enzymes (BACE1), and matrix metalloproteases (MMPs). ADAM17 (A Disintegrin and A Metalloproteinase 17; also known as Tumor necrosis factor-α Converting Enzyme, TACE) was the first sheddase identified and was initially described to be responsible for releasing soluble Tumor Necrosis Factor α (TNFα) from the cell membrane [1,2]. At the moment, more than 30 ADAM17-substrates were described, among them TNFα and its receptors, some EGF-ligands and their receptors, Notch, IL15Rα and Interleukin-6 Receptor (IL6R) [3,4]. Ectodomain shedding has a significant impact on the biological functions of these proteins by converting them from membrane- into Abbreviations: ADAM, a disintegrin and metalloprotease domain; CTF, carboxylterminal fragment; EGF, epidermal growth factor; ICD, intracellular domain; GFP, green fluorescent protein; MEF, mouse embryonic fibroblast; MVB, multivesicular body; PMA, phorbol myristate acetate; RIP, regulated intramembrane proteolysis; WT, wild type ⁎ Corresponding author. Institute of Biochemistry, Christian-Albrechts-University of Kiel, Olshausenstr. 40, D-24118 Kiel, Germany. Tel.: +49 431 880 3336; fax: +49 431 880 5007. E-mail address: [email protected] (S. Rose-John). 0167-4889/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2009.12.001

soluble acting molecules. In addition, the loss of receptors from the cell membrane may lead to the loss of responsiveness of the cells to their cognate ligands [3,5]. In humans, the soluble IL6R can be generated by alternative splicing and by ectodomain shedding [6,7]. We have shown previously that different stimuli such as phorbol 12-mirystate 13acetate (PMA), bacterial toxins, bacterial metalloproteinases and apoptosis led to the cleavage of the IL6R [8–10]. Induced ectodomain shedding of the IL6R is mainly mediated by ADAM17 and constitutive shedding is mediated by ADAM10 [11]. Unlike most soluble receptors, the IL6R does not act as an antagonist, but as an agonist of IL6signaling [12]. We now know that the pro-inflammatory activities of IL6 rely mostly on the sIL6R [13–16]. IL6 mediated signals are important for the coordination of the immune system and IL6 dysregulation can lead to chronic inflammation and cancer development [17,18]. The membrane proteins gp130 and IL6R constitute the signal transducing complex for IL6 stimulation. The agonistic soluble IL6R in complex with IL6 can stimulate cells only expressing gp130 in a process called IL6-trans-signaling [12]. Since most cells of the body do not express the membrane bound IL6R, most cells are normally not responsive to IL6 alone. IL6-transsignaling is important for the regulation of cellular differentiation and apoptosis and has direct consequences on acute and chronic inflammation and on tumor development. All current data imply that IL6-trans-signaling but not classic signaling is the main driving force

A. Chalaris et al. / Biochimica et Biophysica Acta 1803 (2010) 234–245

of chronic inflammation [19]. Therefore, signals via the membrane bound IL6R might only be responsible for normal homeostasis and signals via the sIL6R might rather be important during pathophysiological conditions [13–16]. Ectodomain shedding of transmembrane proteins by so-called αsecretases not only produces extracellular fragments, but also generates intramembrane/intracellular protein stubs, which initially remain in the plasma membrane. Regulated intramembrane proteolysis (RIP) was described as a process how type I or type II-transmembrane proteins are proteolytically processed within the hydrophobic environment of a lipid bilayer after ectodomain shedding [20]. Four known classes of intramembrane cleaving proteases were described: the site-2 protease (S2P), the signal peptide peptidases (SPP), the rhomboids and the presenilins (PS, γ-secretase complex). The γ-secretase cleaves the remaining membrane bound fragment into two peptides, the intracellular domain (ICD) and a small peptide, which is secreted. The γsecretase complex composed of several subunits including nicastrin, APH-1 (anterior pharynx defective 1), PEN-2 (presenilin enhancer-2) and the catalytically active presenilin (PS) 1 or PS2 [21–23]. γ-secretase cleaves numerous type I substrates including Notch and the α-Amyloid Precursor Protein (APP) [21,24,25]. The ectodomain of nicastrin might bind the newly generated amino terminus of APP and Notch domains that were generated upon ectodomain shedding, thereby recruiting APP and Notch intramembrane stubs into the γ-secretase complex [26], however this view has recently been challenged [27]. Cleavage within the hydrophobic membrane might be facilitated in an inner-membrane water-filled cavity constituted by a hydrophobic pore of two secretase transmembrane domains [28,29]. In both cases, the intracellular domains of the substrates are normally released to the cytosol. Whereas the Notch-ICD mediates signaling, a functional role of the APP-ICD is currently under debate [21,25]. Since a definitive biological function could not be determined for the majority of substrates, the question was raised whether the γ-secretase is the “proteasome of the membrane” [30]. The IL6R is a substrate of ADAM10 and ADAM17, but the remaining C-terminal membrane (CTF) stub resulting from ectodomain shedding of the IL6R was not detected and characterized. Here we show that ADAM10 and ADAM17-mediated cleavage of the IL6R led to CTF production by γ-secretase cleavage. Importantly, the α-cleavage preceded the γ-cleavage. Since we did not detect nuclear translocation of the IL6R-CTF, the γ-cleavage most likely serves to remove the membrane stub of the IL6R after shedding. 2. Materials and methods

235

pMOWS vector was incubated with DNA-Polymerase I Klenow (large) fragment to fill in the sticky ends. The PmeI digested IL6R-2Z fragment was subsequently subcloned into the blunt end opened pMOWS vector. The cDNA for the IL6R-GFP fusion protein was assembled using SOEPCR. The primer pair IL6R-EGFP-up (5′-CAGACTACTTCTTCCCCAGAATGGTGAGCAAGGGCGAGGAG-3′) and EGFP-N1-down (5′GGCTGATTATGATCTAGAGTCG-3′) was used to amplify the GFP gene from pEGFP-N1 (Clontech, Palo Alto, USA); the primer pair IL6RupEcoRV (5′-ACTGGATATCGGGCTGAACGGTCAAAGACAT-3′) and EGFPIL6R-down (5′-CTCCTCGCCCTTGCTCACCATTCTGGGGAAGAAGTAGTCTG-3′) was used to amplify the IL6R fragment from p409-IL6R [31]. DNA fragments were gel purified, combined and used as a template for amplification using the flanking primers IL6Rup-EcoRV and EGFPIL6R-dn. The resulting 500 bp product was subcloned into the EcoRV/ NotI digested pBSK-IL6R vector. After sequencing, the IL6R-GFP fragment was removed with HindIII and NotI and inserted into the multiple cloning site of the HindIII/NotI digested pcDNA3.1 vector. The expression vector pcDNA3.1-IL6R-ICD-GFP was generated by using the DNA from the pcDNA3.1-IL6R-GFP as a template. The PCR product was inserted between HindIII and NotI sites of the pcDNA3.1 vector. The primer pair 1072-ICD-IL6RNotI (5′-GATCGCGGCCGCCGATTCTTCTT CAGTACCAC-3′) and EGFP-dn-BamH1 (5′-GATCGGATCCTTACTTGTACA GCTCGTCC-3′) was used to amplify the IL6R-CTF3-GFP fragment from pcDNA3.1-IL6R-GFP. The resulting 1050 bp product was subcloned into the pcDNA3.1 vector by using the restriction sites NotI and BamHI. 2.2. Reagents The metalloprotease inhibitors GI254023X (ADAM10), GW280264X (ADAM10 and ADAM17) and marimastat were a generous gift from Glaxo Smith Kline (Stevenage, United Kingdom) [32]. 12-myristate 13acetate (PMA), the proteasome inhibitor MG132 and the γ-secretase inhibitors DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine-t-butyl-ester) and L-685,485 were purchased from Calbiochem (Schwalbach am Taunus, Germany). The PKC inhibitors GF10920X (panPKC inhibitor) and Gö6976 (Ca2+-dependent PKC inhibitor) were purchased from Sigma-Aldrich (Deisenhofen, Germany). The monoclonal anti-Protein-A antibody was from Sigma-Aldrich (Deisenhofen, Germany); the polyclonal rabbit anti-phospho-STAT-3 (Tyr-705) antibody as well as the monoclonal mouse anti-STAT-3 antibody were purchased from Cell Signalling Technology (Beverly,USA); horseradish peroxidase-coupled goat anti-rabbit and anti-mouse antibodies were obtained from Pierce Biotechnology (Rockford, USA). The human IL6R specific mAB 14–18 was described elsewhere [15].

2.1. Cloning procedure 2.3. Cell culture The cDNA for the IL6R-2Z fusion protein was assembled using splicing by overlapping extension-polymerase chain reaction (SOEPCR). The primer pair ProteinA-ovp-up (5′-GACTACTTCTTCCCCAGAGTCGAGCTGGTTCCGCGTG-3′) and ProteinA-BamHI-down (5′GGATCCTCCCATGGCTACTTTCGGCGCCTGAGCA-3′) was used to amplify the 2Z cDNA from pQE60–2Z [28]; the primer pair IL6Rup EcoRV (5′ ACTGGATATCGGCTGAACGGTCAAAGACAT-3′) and IL6R-ovp-dn (5′CACGCGGAACCAGCTCGACTCTGGGGAAGAAGTAGTC-3′) was used to amplify the IL6R fragment from p409-IL6R [31]. DNA fragments were gel purified, combined and used as a template for amplification using the flanking primers IL6Rup-EcoRV and ProteinA-BamHI-dn. The resulting 1100 bp product was subcloned into the EcoRV/BamHI digested pcDNA3.1-IL6R vector. The IL6R-2Z fragment was removed with SalI and NotI and subcloned into the multiple cloning site of the SalI and NotI sites of p409. The retroviral expression vector pMOWS-IL6R-2Z was constructed by insertion of the cDNA coding for the IL6R-2Z fusion protein into the pMOWS vector. Briefly, the IL6R-2Z fragment was obtained by digestion of the p409-IL6R-2Z vector with the restriction enzyme PmeI and purified by gel extraction. The BamHI/EcoNI digested

COS-7, HepG2 and HEK293 cells were obtained from the American Type Culture Collection (Rockville, MD/Manassas, VA, USA). Transfected Ba/F3 cells have been described [13–16]. ADAM17-deficient and ADAM17-reconstituted mouse embryonal fibroblasts have been described previously [1]. Phoenix cells were previously described [33]. Murine embryonic fibroblast cell lines deficient for presenilin 1 and 2 (PS1/2) were obtained from Paul Saftig, Institute of Biochemistry, University of Kiel [34]. All cells were grown in DMEM High Glucose Culture Medium (PAA Laboratories, Pasching, Austria) supplemented with 10% fetal calf serum, penicillin (60 mg/l) and streptomycin (100 mg/l) at 37 °C with 5% CO2 in a humidified atmosphere. 2.4. Transfection, transduction and selection of cells HepG2 and COS-7 cells were transiently transfected using DEAE Dextran as previously described [35]. The transfection efficiency as visualized after 24 h by GFP expression (Axiovert 200 Microscope, Zeiss) was approximately 60%.

236

A. Chalaris et al. / Biochimica et Biophysica Acta 1803 (2010) 234–245

Mouse embryonic fibroblasts (MEFs) were retrovirally transduced. Transduction of mammalian cells with the retroviral vector pMOWS was been described previously [33]. Briefly, transfection of the Phoenix-Eco packaging cell line with pMOWS-IL6R-2Z using Lipofectamin 2000 (Invitrogen, Karlsruhe, Germany) was done according to the manufacturer's instructions. The cell supernatant containing the retroviruses was collected 24 h post-transfection and filtered through a 0.45 µm filter. For transduction, 500 µl of retroviral supernatant was supplemented with 1.5 µg/ml polybrene and was added to 1×105 MEF cells and centrifuged at 500 ×g at room temperature for 2 h. Afterwards the retroviral supernatant was removed and replaced with standard cell culture medium. For selection of stably transfected MEFs 48 1 h post-transduction 2 µg/ml puromycin was added to the cell culture medium. 2.5. Immunoblotting and enhanced chemiluminescense (ECL) detection For Western blotting proteins, separated by SDS-PAGE were transferred to polyvinylidendifluoride membranes by a semidry electroblotting procedure. Membranes were blocked in a solution of TBS (10 mM Tris, pH 8, 150 mM NaCl) supplemented with 0.02% Tween and 5% skimmed milk powder for at least 1 h, probed with indicated antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were detected by chemiluminescence using the enhanced chemiluminescence kit (Amersham Biosciences Inc.) following the manufacturer's instructions. Signals were quantified using the ImageJ software (http://rsbweb.nih.gov/ij/). 2.6. ELISA IL6R-ELISA was performed as previously described [15]. Microtitre plates (Greiner Microlon, Solingen, Germany) were coated with antihuman IL6R MAB 227 (R&D Systems, Wiesbaden Germany) in PBS. After blocking with PBS supplemented with 5% BSA, 100 µl aliquots of culture supernatants and standards (rhIL6R; R&D Systems) were added as a dilution in PBS supplemented with 1% BSA. IL6R bound to the plate was detected by biotinylated goat anti-human IL6R antibody BAF 227 (R&D Systems) followed by streptavidin-horseradish peroxidase (R&D Systems). The enzymatic reaction was performed with soluble peroxidase substrate (BM blue POD from Roche, Mannheim, Germany) at 37 °C and the absorbance was read at 450 nm on a SLT Rainbow plate reader (Tecan, Maennedorf, Switzerland). 2.7. Membrane preparation and immunoprecipitation Isolation of cellular membranes and immunoprecipitation of the IL6R in supernatants and cell lysates followed by Western blotting was performed as described before [32]. 2.8. Immunocytochemistry HEK293 cells were transfected with pcDNA3.1-IL6R-GFP, pcDNA3.1IL6R-ICD-GFP or with the control plasmid pcDNA3.1-GFP using Lipofectamin 2000 (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. 24 h post-transfection the cells was plated on polylysine-coated glass coverslips. After 24 h the cells were stimulated with PMA and afterwards immediately fixed in 4% paraformaldehyde (Carl Roth, Karlsruhe, Germany) for 15 min at room temperature. Following two washing steps with PBS, the cells were permeabilized with PBS containing 0.1% Triton X-100 for 30 min. After permeabilization, the cells were incubated with DAPI-solution (50 µg/ml DAPI solved in PBS) for 15 min at room temperature. After two washing steps, cells were rinsed thoroughly and mounted in slow Fade anti-Fade medium (Molecular Probes, Göttingen Germany). For LAMP-1 immunocytochemistry, HEK293 cells were transfected and

stimulated as described above and fixed in 4% paraformaldehyde (Carl Roth, Karlsruhe, Germany) for 15 min at room temperature. Following two washing steps with PBS, cells were permeabilized with PBS containing 0.2% saponin for 5 min. After 2 washing steps in PBS, the cells were incubated in glycine buffer for 10 min (0.2% saponin, 0.12% glycine in PBS), washed two times in PBS followed by one blocking step (3% BSA, 0.2% saponin in PBS) for 1 h. Afterwards, cells were incubated with the rat anti-mouse LAMP-1 antibody (clone 1D4B, Developmental Studies Hybridoma Bank; Iowa City, IA, USA) in a 1:50 dilution for one hour in antibody buffer (3% BSA, 0.2% saponin in PBS). Cells were washed 3 times with PBS and incubated with the Alexa-594 fluorochrom-conjugated secondary anti-mouse antibody (Invitrogen, Karlsruhe, Germany) in a 1:300 dilution in antibody buffer in the dark. Afterwards, the cells were washed 3 times and were incubated with DAPI-solution (50 µg/ml DAPI solved in PBS) for 15 min at room temperature. After two washing steps in PBS, cells were rinsed thoroughly and mounted in slow fade anti-Fade medium (Molecular Probes, Göttingen Germany). 3. Results 3.1. Identification of IL6R-2Z derived C-terminal fragments Since specific anti-IL6R antibodies could not detect C-terminal IL6R fragments (CTFs) after PMA-induced shedding (data not shown), we wanted to develop a cellular assay using C-terminal IL6R fusion proteins to monitor the generation of C-terminal fragments of the IL6R in cell lysates of transfected cells after α-secretase cleavage. This method was successfully employed to detect C-terminal fragments of amyloid precursor (APP) [36] and APP protein gene family members APLP-1 and APLP-2 [37] and the membrane bound chemokines CX3CL1 and CXCL16 [38]. The failure to detect natural IL6R C-terminal fragments might be due to rapid degradation of IL6R CTFs as a consequence of its small size. The primary IL6R-CTF after PMA-induced ADAM17-cleavage between Gln357 und Asp358 [10] would consist of 111 amino acid residues. Previous reports have documented the usefulness of the 2Ztag from Staphylococcus aureus Protein-A and GFP for the characterization of CTFs derived by proteolytical processing of the members of the Amyloid Precursor Protein family [37,39]. 2Z-tagged proteins could be visualized on immunoblots using anti-Protein-A monoclonal antibodies. For life cell imaging and to verify the protein cleavage pattern of IL6R-2Z fusion protein, we generated an IL6R-GFP fusion protein. The C-terminus of the IL6R was genetically fused to the 2Z domain of Protein-A consisting of two IgG-binding domains or to GFP (Fig. 1A). An expression plasmid encoding the IL6R-2Z-cDNA was transfected in COS7, HepG2 or HEK293-cells to analyze the expression pattern of IL6R-2Z. As a control, cells transfected with an EGFPexpression plasmid were used. Upon PMA treatment, the extracellular portion of the IL6R-2Z fusion protein or the IL6R-GFP fusion protein (Fig. 1B) was efficiently released from the cell surface. The extracellular portion of the IL6R-2Z fusion protein together with IL6 was as active as the wild type sIL6R protein in stimulating the phosphorylation of STAT3 in Ba/F3 cells stably transfected with a gp130 cDNA (Fig. 1C). In Western blot analysis of cell lysates using an anti-Protein-A antibody, the full length IL6R-2Z-protein was detected as a single band with an apparent molecular weight of ∼ 120 kDa. A second protein of about 115 kDa likely reflects immature IL6R with an incomplete glycosylation pattern. Four additional proteins in the molecular range between 40 and 30 kDa were detected, which probably were CTFs of the IL6R (Fig. 1D, E). No bands were detected in lysates from the EGFP-transfected control cells (Fig. 1D). When the same lysates were examined by immunoblotting with the monoclonal anti-IL6R antibody 14–18, which recognizes an epitope in the ectodomain of the IL6R, only the ∼ 120 and 115 kDa bands were detected in cells transfected with the IL6R-2Z cDNA but not in lysates

A. Chalaris et al. / Biochimica et Biophysica Acta 1803 (2010) 234–245

237

Fig. 1. IL6R-derived C-terminal fragments. (A) Schematic representation of the IL6R-2Z and IL6R-GFP fusion proteins. Numbers indicate positions of the first amino acid residue for each domain. (B) HepG2 cells were transiently transfected with expression plasmids coding for IL6R, the fusion protein IL6R-2Z or Hek293 cells were transiently transfected with expression plasmids coding for the fusion protein IL6R-GFP. The cells transiently transfected with IL6R-2Z cDNA were stimulated with PMA (100 nM) for 2 h or left untreated and cells transiently transfected with IL6R-GFP cDNA were stimulated with PMA (100 nM) for 30 min or left untreated. The amounts of sIL6R in cell culture supernatants were quantified by ELISA and normalized on fold-induction after PMA treatment. (C) HepG2 cells were transiently transfected with expression plasmids coding for IL6R or IL6R-2Z. The cells were stimulated with PMA for 2 h or left untreated. The supernatants of HepG2 cells expressing the IL6R or IL6R-2Z were supplemented with 200 ng/ml IL6. After an incubation period of 30 min the supernatants were used for stimulation of Ba/F3-gp130 cells for 10 min. Ba/F3-gp130 cells were immediately lysed and the proteins were immunoblotted with an anti-P-STAT3 antibody. Re-probing with an anti-STAT3 specific antibody confirmed comparable protein loading. (D) HepG2 cells were transiently transfected with expression plasmids coding for EGFP or IL6R-2Z. After 48 h cell lysates were prepared and analyzed by Western blotting using anti-Protein-A antibody. (E) COS-7 and HepG2 cells were transiently transfected with an expression plasmid coding for IL6R-2Z. The cells were incubated with or without PMA and analyzed by Western blotting using anti-Protein-A mAB for detection of full length IL6R-2Z and CTFs thereof. (F) HepG2 cells were transiently transfected with a plasmid encoding IL6R-GFP (two right lanes) or with a mock plasmid (left lane). After 48 h the cells were stimulated with PMA (100 nM, 2 h). Cell lysates were prepared and analyzed by Western blotting using anti-GFP antibody. (G) HepG2 cells were transiently transfected with an expression plasmid coding for IL6R-2Z. Cells were incubated for 5 min with PMA and then the medium was exchanged against fresh medium lacking PMA. The cells were harvested after indicated time points and analyzed by Western blotting using anti-Protein-A mAB for detection of CTF3.

238

A. Chalaris et al. / Biochimica et Biophysica Acta 1803 (2010) 234–245

from EGFP-transfected cells (data not shown), again suggesting that the four smaller fragments detected by the anti-Protein-A antibody represented IL6R CTFs. We therefore termed the corresponding polypeptide species as CTF1 (∼ 40 kDa), CTF2 (∼ 38 kDa), CTF3 (∼33 kDa) and CTF4 (∼ 30 kDa). Interestingly, the calculated mass of the C-terminal IL6R-2Z-fragment occurring after ADAM17-induced cleavage was calculated to be 32.7 kDa, which closely reflects the size of CTF3 (compare Fig. 1E). 3.2. Induction of CTF3 formation by PMA treatment of cells It is well established that shedding of the IL6R ectodomain can be stimulated by the phorbol ester PMA, which exerts its effect via activation of protein kinase C (PKC). Treatment of cells expressing IL6R-2Z and IL6R-GFP with 10− 7 M PMA led to a massive release of the sIL6R (Fig. 1B). We therefore reasoned that PMA treatment of cells transiently transfected with a cDNA coding for IL6R-2Z might influence the formation of IL6R derived CTFs. Interestingly, treatment of HepG2 cells transiently transfected with IL6R-2Z cDNA with PMA for 2 h selectively increased the level of CTF3 in cell lysates. Comparable results were also obtained in COS-7 cells transiently transfected with IL6R-2Z cDNA, thus demonstrating that the observed induction of CTF3 formation was not cell-type dependent (Fig. 1E). Experiments with cells transiently transfected with IL6R-GFP cDNA yielded the same results (Fig. 1F). We also found a slight PMAdependent upregulation of CTF1/2 and CTF4 in some experiments as shown in Fig. 1F. However, this upregulation could not be reproduced in all experiments. At the moment, we cannot explain the effect of PMA on the formation of CTF2 and CTF4. The selective induction of CTF3 by PMA suggested that CTF3 was generated by a protease, possibly ADAM17, which is responsive to activated PKC and which was distinct from the protease(s) responsible for the formation of CTF1, CTF2 and CTF4. The increase of CTF3 was not accompanied by a reduction in band intensity of the full length IL6R, which might be due to over-expression of IL6R-2Z in the transiently transfected cells. To assess the kinetics of CTF3-formation following PMA treatment of cells, IL6R-2Z expressing HepG2 cells were treated with 10− 7 M PMA for 5 min and thereafter the medium was exchanged for fresh medium lacking PMA. At time points indicated in Fig. 1G, cells were harvested, lysed and the CTF3 levels were monitored over a period of 8 h by immunoblotting with the anti-Protein-A antibody. A significant increase of the CTF3 fragment intensity was evident already 1 min after PMA stimulation, reached a maximum between 15 and 30 min and thereafter decreased to background level after 8 h. This result indicated that the PMA-induced CTF3 formation is a rapid process, which might suggest the involvement of the metalloproteinase ADAM17 [7]. 3.3. IL6R-CTF3 is generated by ADAM17 It was shown previously that constitutive cleavage of the IL6R is mediated by ADAM10, whereas ADAM17 is responsible for PMAinduced cleavage [11]. HepG2 cells transiently transfected with IL6R-2Z cDNA were treated with PMA in the presence of two hydroxamate based inhibitors, which were previously demonstrated to differentially block ADAM10 and ADAM17 [40]. The inhibitor GW280264X (GW) blocked both, ADAM10 and ADAM17, whereas GI254023X (GI) preferentially blocked ADAM10 [15,32]. The effect of the broad spectrum metalloproteinase inhibitor marimastat on CTF formation was also examined [15,32]. None of the mentioned inhibitors affected the formation of CTF1, CTF2 and CTF4 (data not shown). As described before, treatment of the cells with PMA induced the production of CTF3 (Fig. 2A). The amount of the IL6R-CTF3 fragment was reduced following treatment of cells with PMA in the presence of the preferential ADAM10-inhibitor GI254023X (Fig. 2A) whereas the ADAM10/17 inhibitor GW280264X completely blocked the formation of CTF3, thus confirming that this

fragment is generated with the help of ADAM17 (Fig. 2A). The IL6Rcleavage site used by the protease ADAM10 has not been defined at the molecular level; since both inhibitors suppressed the formation of the same CTF, our results indicate that ADAM17 and ADAM10 use the same or adjacent cleavage sites within the IL6R. Examination of corresponding conditioned media confirmed that GW280264X suppressed PMA induced release of the IL6R ectodomain into the cell culture medium, whereas only a minor inhibition of PMAinduced IL6R-2Z shedding was seen after treatment with the preferential ADAM10 inhibitor GI254023X (Fig. 2B). Of note, none of the inhibitors influenced the levels of CTF1, CTF2 and CTF4, raising the question whether these proteins might reflect unspecific degradation products of the IL6R-2Z-fusion protein (data not shown and Fig. 4D). Next, we used ADAM17-deficient murine embryonic fibroblasts (MEFs) and ADAM17-deficient MEFs reconstituted with a cDNA coding for ADAM17. These cells were retrovirally transduced with an IL6R-2ZcDNA and were treated with PMA for 2 h or were left untreated. PMAinduced shedding of the IL6R was almost completely abolished in ADAM17-deficient MEFs, whereas the ADAM17-reconstituted MEFs displayed normal PMA-induced IL6R shedding (Fig. 2C). As depicted in Fig. 2D, CTF3 could only be detected in MEFs reconstituted with an ADAM17 cDNA but not in ADAM17-deficient MEFs. Interestingly, the overall occurrence of CTF1/2 (one band) but not of CTF4 was reduced in the ADAM17-deficient MEFs as compared to the ADAM17 reconstituted MEFs in the presence and absence of PMA. The different CTF1/2 levels could be related to the independent clonal origin of the MEFs. Importantly, even though full length IL6R protein could be detected in both cell lines at comparable amounts, no CTF3 could be detected after PMA stimulation in the absence of ADAM17, indicating that CTF3 is generated by ADAM17-mediated cleavage of the IL6R-2Z protein. Since the PMA-stimulated generation of CTF3 was transient and the detection level varied in different cell lines, we concluded that CTF3 was rapidly degraded after generation. 3.4. Ca2+-dependent PKCs are involved in the PMA-induced CTF3 generation PMA-induced ectodomain shedding of diverse membrane proteins such as IL6R is induced via activation of protein kinases of the PKC family. Using the PKC inhibitors GF109203X and Gö6976 we analyzed whether members of the PKC family are involved in the PMA-elicited increase in CTF3 formation. HepG2 cells transiently transfected with the IL6R-2Z-cDNA were treated with the PKC inhibitors alone or in combination with PMA and the formation of the IL6R-2Z derived CTF3 was examined by immunoblotting (Fig. 2E). PMA-induced CTF3 formation was suppressed by the pan-PKC kinase inhibitor GF109203X and the inhibitor of Ca2+-dependent PKC isoenzymes Gö6976. Examination of sIL6R levels in conditioned medium with an antibody recognizing an epitope in the ectodomain of sIL6R confirmed that the PMA-induced release of the sIL6R was suppressed in the presence of both inhibitors (Fig. 2E). Thus, this experiment suggested the involvement of Ca2+-dependent PKC isoenzymes as mediators of PMA-induced stimulation of IL6R-CTF3 formation. 3.5. The CTF3 of IL6R is a substrate of the γ-secretase Regulated intramembrane proteolysis is a process, by which transmembrane proteins are proteolytically cleaved within their transmembrane domains by a protease complex termed γ-secretase usually preceded by an α-secretase (ADAM10 or ADAM17) mediated cleavage within the ectodomain. This process leads to the generation of intracellular domains (ICDs) from the previously generated C-terminal fragments (CTFs). DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-Sphenylglycine-t-butyl-ester) was described to inhibit γ-secretase activity via binding to the C-terminal fragment of presenilin [41]. To address whether the IL6R represents a novel substrate for the γ-

A. Chalaris et al. / Biochimica et Biophysica Acta 1803 (2010) 234–245

239

Fig. 2. CTF3 is generated by ADAM17 after PMA treatment. (A) HepG2 cells were transiently transfected with an expression plasmid coding for IL6R-2Z and stimulated for 2 h with and without PMA (100 nM) in the presence and absence of the protease inhibitors GW280264X (GW, 3 µM) and GI254023X (GI, 3 µM). Cells were harvested and analyzed by Western blotting using anti-Protein-A mAB for detection of CTF3. (B) HepG2 cells were transiently transfected with an expression plasmid coding for IL6R-2Z and stimulated for 2 h with and without PMA in the presence and absence of the protease inhibitors GW280264X (GW) and GI254023X (GI). The sIL6R protein in the culture media was quantified using a sIL6R-specific ELISA. (C) IL6R-2Z-cDNA retrovirally transduced ADAM17-deficient MEFs and reconstituted MEFs were treated with PMA for 2 h. The sIL6R protein in the culture media was quantified using a sIL6R-specific ELISA. (D) IL6R-2Z-cDNA-stably transduced ADAM17-deficient MEFs and reconstituted MEFs were treated with PMA for 2 h. The occurrence of IL6R-CTF3 in cell lysates was assessed by Western blotting using anti-Protein-A mAB. (E) HepG2 cells were transiently transfected with an expression plasmid coding for IL6R-2Z and stimulated for 2 h with and without PMA in the presence and absence of the PKC inhibitors GF109203X (GF, 5 µM) and Gö6976 (Gö, 1 µM). The cells were harvested and analyzed by Western blotting using anti-Protein-A mAB for detection of CTF3. The sIL6R protein in the culture media was immunoprecipitated and analyzed by Western blotting using anti-IL6R mAB for detection of sIL6R.

secretase complex, HepG2 cells transiently transfected with IL6R-2Z cDNA were treated with DAPT in a dose-dependent manner over a concentration range of 0.1 to 10 µM DAPT in the presence or absence of PMA and cell lysates were examined by immunoblotting (Fig. 3A). Whereas PMA treatment alone only led to a faint CTF3 protein fragment formation, combined treatment with PMA and DAPT resulted in considerably more CTF3 formation. The results were quantified by density calculation of CTF3 using the ImageJ software. A dosedependent PMA-dependent and independent increase of CTF3 became apparent already after the treatment of the cells with 0.1 µM DAPT, however this effect was maximal after treatment of the cells with 5– 10 µM DAPT. Additionally, we have used the γ-secretase inhibitor L-685,485. Dose–response experiments were performed from 0.1 µM to 5 µM L685,485 (Fig. 3B). The results were quantified by density calculation of CTF3 using the imageJ software. A dose-dependent increase of CTF3 became apparent already after the treatment of the cells with 0.5 µM L-685,485, however this effect was maximal after treatment of the cells with 5 µM L-685,485. Next, ADAM17-deficient MEFs and ADAM17-reconstituted MEFs retrovirally transduced with IL6R-2Z cDNA were treated with DAPT in the presence or absence of PMA for 2 h. Again, CTF3 specifically accumulated in PMA treated ADAM17-reconstituted MEFs but not in ADAM17-deficient MEFs. Accumulation of CTF3 was further increased by combined PMA and DAPT treatment in ADAM17-reconstituted MEFs and in ADAM17 deficient MEFs, indicating that processing of CTF3 is mediated by the γ-secretase complex (Fig. 3C). The minor accumulation of the CTF3 after PMA and DAPT treatment in the ADAM17-deficient MEFs indicated that CTF3 generated by other

proteases such as ADAM10 was also a substrate of the γ-secretase complex. We detected an increased intensity of the CTF4 fragment (Fig. 3A, B and C), which might indicate that also CTF4 is a substrate of the γ-secretase. Since the increase in CTF4 was not seen in all experiments, we cannot exclude that CTF4 might reflect an unspecific degradation product of the IL6R-2Z-fusion protein. To further corroborate that the PMA-induced CTF3 of the IL6R is a novel substrate of the γ-secretase complex, we used MEFs deficient in the catalytically active γ-secretase subunits presenilin (PS)1 and PS2, which were transduced with IL6R-2Z cDNA. Although wild type MEFs showed less expression of the IL6R-2Z protein as compared to the PS1/PS2-deficient MEFs, the ratio of the secreted IL6R after PMA stimulation showed only minor differences between both cell types, indicating that α-secretase cleavage is not affected by γ-secretase deficiency (Fig. 4A). Next, we investigated the cell lysates for the presence of IL6R fragments. Again, PMA-induced CTF3 formation was only faintly visible in wild type cells whereas a predominant band corresponding to CTF3 was detected in PS1/PS2-deficient MEFs (Fig. 4B). Taken together these data showed that the IL6R-CTF3 is a novel substrate of γ-secretase-mediated regulated intramembrane proteolysis. To further confirm the effect of γ-secretase inhibition on the fate of the IL6R, HepG2-cells transiently transfected with IL6R-2Z cDNA were treated with PMA in the presence or absence of DAPT. The cells were lysed and divided into membrane and cytosolic fractions (Fig. 4C). No CTF3-fragment could be detected in the cytosolic fraction. As expected, all IL6R-CTFs including CTF3 were found in the membrane fraction, at least until cleavage by the γ-secretase had occurred (Figs. 4C and 5B). The cleavage of the IL6R by ADAM10/17 occurred in

240

A. Chalaris et al. / Biochimica et Biophysica Acta 1803 (2010) 234–245

Fig. 4. Accumulation of IL6R-CTF3 in γ-secretase deficient cells. (A) PS1/2-deficient MEFs and reconstituted MEFs, stably transduced with IL6R-2Z-cDNA were treated with PMA for 2 h. The occurrence of sIL6R was assessed by ELISA. (B) PS1/2-deficient MEFs and wild type (WT) MEFs, stably transduced with IL6R-2Z-cDNA were treated with PMA for 2 h. The occurrence of CTF3 was assessed by Western blotting using anti-Protein-A mAB. (C) HepG2 cells were transiently transfected with an expression plasmid coding for IL6R-2Z and were treated with PMA for 2 h in the presence or absence of the γ-secretase inhibitor DAPT. The occurrence of CTF3 in the membrane [m] or cytosolic [c] fraction was assessed by Western blotting using anti-Protein-A mAB. (D) HepG2 cells were transiently transfected with an expression plasmid coding for IL6R-2Z and stimulated for 24 h with and without the proteosome inhibitors MG132 (MG, 2.5 µM), lactacystin (L, 15 µM) or the metalloprotease inhibitors GW280264X (GW, 3 µM) and GI254023X (GI, 3 µM). The occurrence of the IL6R fusion protein was assessed by Western blotting using anti-ProteinA mAB. Fig. 3. Inhibition of γ-secretase activity leads to accumulation of IL6R-CTF3. (A) HepG2 cells were transiently transfected with an expression plasmid coding for IL6R-2Z and stimulated for 24 h with increasing concentrations of the γ-secretase inhibitor DAPT (0.1–10 µM, as indicated). PMA was added for the last two hours of DAPT treatment. Cells were harvested and analyzed by Western blotting using anti-Protein-A. (B) HepG2 cells were transfected as described in (A) and stimulated for 24 h with increasing concentrations of the γ-secretase inhibitor L-685,458 (0.1–5 µM, as indicated). PMA was added for the last two hours of L-685,458 treatment. Cells were harvested and analyzed by Western blotting using anti-Protein-A. (C) ADAM17-deficient MEFs and reconstituted MEFs, stably transduced with IL6R-2Z-cDNA were treated with PMA for 2 h in the presence or absence of the γ-secretase inhibitor DAPT (10 µM). The occurrence of CTF3 was assessed by Western blotting using anti-Protein-A mAb.

the extracellular portion of the IL6R protein in close proximity to the transmembrane region and the expected cleavage site of the γsecretase would be within the transmembrane region of IL6R. It is generally assumed that the ICD of a γ-secretase substrate is 2–3 kDa smaller than its CTF and might be released into the cytoplasm where it is either transported into the nucleus or rapidly degraded by the proteasome [42,43]. Therefore, the ICD of IL6R-CTF3 would have an

A. Chalaris et al. / Biochimica et Biophysica Acta 1803 (2010) 234–245

241

Fig. 5. IL6R-derived CTF3 is processed by γ-secretase at the plasma membrane and in endosomal compartments. (A) HEK293 cells were transiently transfected with an expression plasmid coding for IL6R-GFP and pulse-stimulated for 5 min with PMA, washed and incubated in cell culture medium for the indicated time points. Cells were fixed in 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton-X100 and GFP-fluorescence was analyzed using the fluorescence microscope Zeiss Axiovert 200M. The nuclei were visualized by DAPI staining; a–f: IL6R-GFP staining, g–l: IL6R-GFP and DAPI staining. (B) HEK293 cells were transiently transfected with an expression plasmid coding for IL6R-GFP and pulse-stimulated for 5 min with PMA in the presence and absence of the protease inhibitor GW280264X (3 µM) and the γ-secretase inhibitor DAPT (10 µM). Cells were washed and incubated in cell culture medium for additional 30 min in the presence and absence of the inhibitors. Cells were fixed in 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton-X100 and GFP-fluorescence was analyzed using the Fluorescence microscope Zeiss Axiovert 200M. The nuclei were visualized by DAPI staining. (C) HEK293 cells were transiently transfected with an expression plasmid coding for the full length IL6R-GFP or an expression plasmid coding for the IL6R-ICD-GFP or GFP alone. After transfection the cells were stimulated for 30 min with PMA (100 nM) or left untreated. The nuclear export inhibitor Leptomycin B (40 nM) was added 4 h prior to PMA stimulation. Cells were prepared and additionally stained with DAPI and for LAMP1.

estimated molecular weight of 30–31 kDa and might be covered by the CTF4 band. Indeed, in some experiments (Fig. 3A and B) we could detect an increased intensity of the CTF4 fragment. We also were not able to detect a cytosolic IL6R-ICD in the soluble fraction. To characterize the degradation pathway involved in the clearance of the IL6R-CTF3/ICD we incubated IL6R-2Z overexpressing HepG2 cells for 24 h with two different proteasome inhibitors. The widely used compound MG132 blocks the proteasome and additional proteases like the lysosomal cathepsins and non-lysosomal calpains

[44]. Lactacystin is supposed to be a specific proteasome inhibitor. Treatment of HepG2 cells with MG132 led to a strong accumulation of the full length IL6R-2Z fusion protein as well as CTF1 and CTF3. In contrast, we did not observe such an accumulation by treating the cells with the more specific proteasome inhibitor lactacystin indicating that the proteasome is not involved in the degradation process of the IL6R CTFs (Fig. 4D). We conclude from these experiments showing that blockade of γ-secretase led to a strong accumulation of IL6R-CTF3 that IL6R-CTF3 is processed by γ-secretase.

242

A. Chalaris et al. / Biochimica et Biophysica Acta 1803 (2010) 234–245

3.6. IL6R-CTF3 is processed by the γ-secretase at the plasma membrane We used HEK293-cells transiently transfected with the GFPtagged IL6R-GFP cDNA to monitor IL6R processing by cellular imaging. The cells were pulse-treated with PMA for 5 min, and occurrence of GFP-fluorescence was monitored for 720 min. Since we previously showed that CTF3 generation was maximal 30–60 min after PMA treatment (Fig. 1G), we decided to use the 30 min time point to follow the fate of IL6R-CTF. The IL6R-GFP fusion protein could be clearly detected at the cell membrane with some GFP-fluorescence being localized in endosomal compartments (Fig. 5A). Nuclei were stained with DAPI to illustrate that the IL6R-CTFs were not accumulating within the nucleus. Almost all fluorescence disappeared from the cell membrane 30 min after the PMA-pulse and it took 360 min for the IL6R-GFP to be re-expressed at the cell membrane. Fig. 5B shows that the loss of PMA-induced IL6R-GFP-fluorescence from the cell surface was blocked by the ADAM10/ADAM17 specific inhibitor GW280264X, indicating that the disappearance of GFP-fluorescence from the plasma membrane was initiated by ADAM17-mediated ectodomain shedding of the IL6R. It was previously shown that γ-secretase may cleave its substrates at the plasma membrane as well as in endosomal vesicles, but not in the ER, the Golgi and in constitutive secretory vesicles [39]. Therefore, HEK293-cells transiently transfected with IL6R-GFP cDNA were treated with PMA in the presence of the γ-secretase inhibitor DAPT (Fig. 5B). Even though ADAM17-mediated ectodomain shedding of the IL6R occurred normally (also compare Fig. 4A), in the presence of DAPT, the IL6R-GFP-fluorescence did not disappear from the cell membrane. Furthermore, we investigated whether the γ-secretase cleavage product of IL6R translocated to the nucleus. We stimulated HEK293 cells transiently transfected with the full length IL6R-GFP cDNA with PMA in the presence of Leptomycin B (LMB), which is an inhibitor of nuclear export. Neither PMA alone or combined PMA and LMB treatment led to visible accumulation of the IL6R-CTF in the nucleus as judged by fluorescence microscopy and DAPI co-staining. Instead, we noted that the GFP-fluorescence after PMA treatment was shifted from the plasma membrane to intracellular structures, probably endosomal vesicles, indicating that the unidentified γ-secretase cleavage product of IL6R remained associated with membraneous structures (Fig. 5C). We genetically engineered a GFP-tagged IL6R-ICD cDNA, which codes for the complete intracellular domain of the IL6R without the transmembrane domain. We then analyzed whether this fragment corresponding to the complete intracellular domain of the IL6R is also associated with endosomal/lysosomal compartments. HEK293 cells were transiently transfected with this GFP-tagged IL6R-ICD cDNA and GFP-fluorescence was analyzed by fluorescence microscopy with and without DAPI costaining. As shown in Fig. 5C, the GFP-tagged IL6R-ICD was mainly detected in the cytosol, but not in endosomal/lysosomal compartments and therefore resembled the situation in cells that were transfected with GFP alone (Fig. 5C, right panel). Treatment of these cells with Leptomycin B also did not lead to an accumulation of the IL6R-ICDGFP in the nucleus. These experiments indicate that IL6R-ICD was not transported to the nucleus. As a control, cells were stained for the late endosomal/lysosomal marker LAMP-1 (see below). To identify the nature of the GFP-fluorescence containing structures, we treated IL6R-GFP overexpressing HEK293 cells with or without PMA and again stained the cells for LAMP-1 (Fig. 6). Under both conditions we found co-localization of IL6R-GFP within the same endosomal structure as the lysosomal protein LAMP-1, which appear in yellow. However, in PMA treated cells we found more colocalization signals within intracellular compartments than in untreated cells (Fig. 6j, l). The increase of these vesicles happened at the same time when IL6R-CTF3 was generated and further processed by the γ-secretase suggesting that these intracellular compartments are filled with IL6R-CTFs and cleavage products thereof.

Fig. 6. γ-secretase cleavage product of IL6R is located in endosomal structures. HEK293 cells were transiently transfected with an expression plasmid coding for the IL6R-GFP. After transfection the cells were left untreated or stimulated for 30 min with 100 nM PMA. Cells were fixed in 4% paraformaldehyde and stained with DAPI and for LAMP1. GFP- and LAMP1-fluorescence was analyzed using the fluorescence microscope Zeiss Axiovert 200M. a–d: GFP staining; e–h: LAMP-1 staining; i–l: merged images. The higher magnification images (b, d) show GFP-fluorescence and LAMP1 fluorescence (f, h) whereas the images (j, l) depict the co-localization of the γ-secretase cleavage product of IL6R-GFP and LAMP-1 in yellow.

3.7. Expression of IL6R-CTF3 leads to intravesicular localization To analyze, whether the ADAM17-dependent increase of intracellular staining in IL6R-GFP transfected cells might be caused by the generation and processing of IL6R-CTF3, we have generated a CTF3mimicking protein. This fusion protein was named IL6R-CTF3-GFP and contained the IL6R signal peptide followed by a c-myc-tag and the Cterminal fragment starting with asp 358 of the human IL6R (Fig. 7A). ADAM17 cleaves the IL6R between gln 357 and asp 358 [10], the natural remaining IL6R cleavage product starts with asp 358. Therefore this construct mimics the IL6R-C-terminal fragment after ADAM17-mediated cleavage. The cDNA coding for IL6R-CTF3-GFP was transiently transfected in HEK293 cells. Upon transfection, the IL6RCTF3-GFP fusion protein was detected as a 37 kDa band upon SDSPAGE and Western blotting (data not shown). We performed fluorescence microscopy using this fusion protein under non-stimulated and PMA-stimulated conditions (Fig. 7B). In transfected cells the fusion protein was localized intracellularly and associated with intracellular membrane structures. Localization of GFP-fluorescence was reminiscent of the GFP-fluorescence of the full length IL6R after PMA-stimulated cleavage. This situation did not change after PMA stimulation of IL6RCTF3-GFP expressing cells. The higher magnification images show colocalization of GFP and LAMP-1 in some vesicles of untreated and PMA treated cells. This situation did not change upon treatment of the transfected cells with Leptomycin B (right panels). These data demonstrate that in most cells an IL6R-CTF3 mimicking fusion protein was translocated to intracellular vesicles. It is noteworthy, that the IL6RCTF3-GFP protein without PMA treatment shows a similar intracellular behavior as the cleaved IL6R-GFP fusion protein upon PMA stimulation of the cells, underlining the fact that this protein construct mimics the cleaved membrane C-terminal fragment of the shed IL6R. 4. Discussion The IL6R exists as a transmembrane and soluble protein. The human sIL6R can be generated by alternative splicing and ectodomain shedding

A. Chalaris et al. / Biochimica et Biophysica Acta 1803 (2010) 234–245

243

Fig. 7. IL6R-CTF3 is located in endosomal structures. (A) Schematic representation of the IL6R-CTF3-GFP fusion protein. (B) HEK293 cells were transiently transfected with an expression plasmid coding for IL6R-CTF3-GFP. After 48 h, cells were stimulated for 30 min with PMA or left untreated. The nuclear export inhibitor Leptomycin B (40 nM) was added 4 h prior to PMA stimulation. Cells were fixed in 4% paraformaldehyde. GFP-fluorescence as well as LAMP-1 and DAPI fluorescence was analyzed using fluorescence microscopy. The higher magnification images show the co-localization of IL6R-CTF3 and LAMP-1 in yellow.

although it is clear that shedding of the IL6R is the predominant mechanism for the generation of the sIL6R protein [6,7,45]. The sIL6R in combination with IL6 can activate cells that do not express membrane bound IL6R but the receptor chain gp130, which is ubiquitously expressed. This process was named IL6-trans-signaling and is important for cellular activation and differentiation [12]. It has been shown that IL6-trans-signaling but not IL6-classic signaling via the membrane bound IL6R is the main driving force of chronic inflammatory states like rheumatoid arthritis and Crohn's disease [19]. Nothing is known about the fate of the C-terminal part of the IL6R, which remains in the membrane after ectodomain shedding. In previous studies we and others demonstrated that in a slow process the IL6R is constitutively released by ADAM10, whereas inducible release triggered e.g. by PMA is a fast process mediated by ADAM17. Sheddases such as ADAM10 and ADAM17 can act in combination with γ-secretase to perform regulated intramembrane proteolysis of the remaining membrane-associated protein and release of ICD. Generation of ICDs followed a specific sequence of proteolytic steps in which ectodomain shedding of the substrate must occur before regulated intramembrane proteolysis of ICD [46].

A number of proteins have been shown to undergo intramembrane proteolysis, including the members of the Amyloid Precursor Protein (APP) family, the receptor tyrosine kinase ErbB4, the lowdensity lipoprotein receptor-related protein (LRP) and the cell adhesion molecules cadherin, CD44 and Notch [47]. In this study we characterized for the first time regulated intramembrane proteolysis of the human IL6R. Cleavage by ADAM17 resulted in the generation of a major 65 kDa soluble IL6R fragment, but the CTF-fragments of the IL6R could not be detected in cell lysates with an antibody specifically directed against the C-terminus of the IL6R. Therefore we decided to construct two C-terminal IL6R fusion proteins IL6R-2Z and IL6R-GFP. 2Z- and GFP fusion proteins were successfully used for the identification and characterization of the CTFs of the type I-transmembrane proteins APP, Notch and APLP1 [39]. Using anti-2Z- and anti-GFP-specific antibodies, four IL6R-CTFs were detected. Activation of ADAM17 resulted in strong accumulation of CTF3 at cellular membranes and in a simultaneous release of the IL6Rectodomain. The generation of CTF3 was almost absent in ADAM17deficient MEFs, which support the finding that CTF3 is generated by ADAM17. The cleavage pattern observed for IL6R-2Z and IL6R-GFP were

244

A. Chalaris et al. / Biochimica et Biophysica Acta 1803 (2010) 234–245

similar, but the abundance of CTF1, CTF2 and CTF4 were not consistently affected by the treatments or in any cell line used in our study. Therefore we concluded that the formation of CTF1, 2 and CTF4 was not related to ADAM17-mediated shedding of the IL6R and was not subject to γsecretase cleavage. Furthermore, we showed that γ-secretase inactivation by inhibitors or the absence of γ-secretase in presenilin 1/2-deficient mouse embryonic fibroblasts resulted in accumulation of CTF3 after PMA stimulation, indicating that only CTF3 was a substrate of the γsecretase complex. Again CTF1, 2 and 4 were not affected. The remaining IL6R-ICD, which is a product of γ-secretase cleavage of CTF3, could not be detected either in the cytosolic or in the membrane fraction of transiently and stably transfected cells. Proteolytic processing of transmembrane proteins is often linked with the release of an ICD into the cytosol which regulates signaling functions of the receptor. Thus, Notch-ICD is known to recruit histone acetyl transferases and RBP-J/CBF-1 to activate gene transcription [48]. In this study we provide experimental evidence for a novel pathway involved in CTF sorting. Even though, we were not able to physically detect an IL6R-ICD, we clearly see a translocation of IL6R-GFPfluorescence into endosomal compartments after ADAM and γsecretase mediated cleavage of the IL6R. Therefore we conclude that the γ-secretase cleavage product of IL6R remained associated with the plasma membrane before it was internalized and transported into endosomal vesicles. A construct, which contained the complete intracellular domain but not the transmembrane domain of the IL6R fused to a GFP-portion (IL6R-ICD-GFP) was not associated with endosomal/lysosomal vesicles. The fluorescence microscopy data support our assumption that after being internalized in endosomal vesicles ICD-GFP might be sorted into internal luminal vesicles of the late endosome. Recently, it was reported, that the cytoplasmic portion of the type I-transmembrane protein HB-EGF after proteolytic processing by ADAM proteases can be transferred to the inner nuclear envelope via endocytosis and early endosomes. A transmembrane-free HB-EGF has never been detected and a membrane bound HB-EGF-cytosolic fragment is a major product after shedding stimuli [49,50]. The endosomal compartments containing the IL6R-ICD-GFP might later fuse with lysosomes where the IL6R-ICD-GFP might be degraded by lysosomal enzymes like cathepsins. However, at this stage, we have not identified the IL6R-ICD nor the proteases involved in IL6R-ICD degradation. So far, we have not identified a physiological role of the IL6R-CTF3. In case of the chemokines CX3CL1 and CXCL16, regulated intramembraneous proteolysis most likely serves as a mechanism to clear the cell membrane from the C-terminal fragments [30]. At the moment, we believe in a similar role of the proteolysis of the IL6R-CTF3 by γsecretase. Since we did not observe IL6R-ICD-GFP-fluorescence within the nucleus, even in the presence of the nuclear export inhibitor Leptomycin B, the IL6R-ICD is most likely not transported into the nucleus and therefore might not be directly involved in gene regulation. Interestingly it was shown that the IL6R-ICD mediates the basolateral sorting of the IL6R in polarized cells. At the present it is unclear, how the cleavage of the IL6R by ADAM17 and its subsequent processing by the γ-secretase interferes with this sorting process and whether and how these processes are coordinated within the cell [51]. It is conceivable that proteolytic processing of the IL6R by γ-secretase, precludes binding to proteins of the intracellular sorting machinery and thereby influences sorting of other proteins. Taken together, our data demonstrate, that the IL6R is rapidly processed by subsequent cleavages mediated by the ADAM17 metalloprotease and γ-secretase, both occurring at the plasma membrane and that this process results in the release of the sIL6R and the degradation of the ADAM17-related C-terminal fragment of the IL6R in intracellular compartments. At the moment we have no indication that a potential IL6R-ICD plays an additional intracellular role before degradation.

Acknowledgements We thank Dr. Paul Saftig for his help with the PS1/2 deficient fibroblasts. We also thank Stefanie Schnell and Michaela Jahn for excellent technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft, Bonn, Germany to J. S. and S. R.-J. (SFB415, Project B5) and by the German Cluster of Excellence ‘Inflammation at Interfaces’. References [1] R.A. Black, C.T. Rauch, C.J. Kozlosky, J.J. Peschon, J.L. Slack, M.F. Wolfson, B.J. Castner, K.L. Stocking, P. Reddy, S. Srinivasan, N. Nelson, N. Boiani, K.A. Schooley, M. Gerhart, R. Davis, J.N. Fitzner, R.S. Johnson, R.J. Paxton, C.J. March, D.P. Cerretti, A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells, Nature 385 (1997) 729–733. [2] M.L. Moss, S.L. Jin, M.E. Milla, D.M. Bickett, W. Burkhart, H.L. Carter, W.J. Chen, W.C. Clay, J.R. Didsbury, D. Hassler, C.R. Hoffman, T.A. Kost, M.H. Lambert, M.A. Leesnitzer, P. McCauley, G. McGeehan, J. Mitchell, M. Moyer, G. Pahel, W. Rocque, L.K. Overton, F. Schoenen, T. Seaton, J.L. Su, J.D. Becherer, Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha, Nature 385 (1997) 733–736. [3] A.P. Huovila, A.J. Turner, M. Pelto-Huikko, I. Karkkainen, R.M. Ortiz, Shedding light on ADAM metalloproteinases, Trends Biochem. Sci. 30 (2005) 413–422. [4] C.P. Blobel, ADAMs: key components in EGFR signalling and development, Nat. Rev. Mol. Cell Biol. 6 (2005) 32–43. [5] J. Arribas, A. Borroto, Protein ectodomain shedding, Chem. Rev. 102 (2002) 4627–4638. [6] S. Horiuchi, Y. Koyanagi, Y. Zhou, H. Miyamoto, Y. Tanaka, M. Waki, A. Matsumoto, M. Yamamoto, N. Yamamoto, Soluble interleukin-6 receptors released from T cell or granulocyte/macrophage cell lines and human peripheral blood mononuclear cells are generated through an alternative splicing mechanism, Eur. J. Immunol. 24 (1994) 1945–1948. [7] J. Müllberg, H. Schooltink, T. Stoyan, M. Gunther, L. Graeve, G. Buse, A. Mackiewicz, P.C. Heinrich, S. Rose-John, The soluble interleukin-6 receptor is generated by shedding, Eur. J. Immunol. 23 (1993) 473–480. [8] P. Vollmer, I. Walev, S. Rose-John, S. Bhakdi, Novel pathogenic mechanism of microbial metalloproteinases: liberation of membrane-anchored molecules in biologically active form exemplified by studies with the human interleukin-6 receptor, Infect. Immun. 64 (1996) 3646–3651. [9] I. Walev, P. Vollmer, M. Palmer, S. Bhakdi, S. Rose-John, Pore-forming toxins trigger shedding of receptors for interleukin 6 and lipopolysaccharide, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 7882–7887. [10] J. Müllberg, W. Oberthur, F. Lottspeich, E. Mehl, E. Dittrich, L. Graeve, P.C. Heinrich, S. Rose-John, The soluble human IL-6 receptor. Mutational characterization of the proteolytic cleavage site, J. Immunol. 152 (1994) 4958–4968. [11] V. Matthews, B. Schuster, S. Schütze, I. Bußmeyer, A. Ludwig, C. Hundhausen, T. Sadowski, P. Saftig, D. Hartmann, K.-J. Kallen, S. Rose-John, Cellular cholesterol depletion triggers shedding of the human interleukin-6 receptor by ADAM10 and ADAM17 (TACE), J. Biol. Chem. 278 (2003) 38829–38839. [12] S. Rose-John, P.C. Heinrich, Soluble receptors for cytokines and growth factors: generation and biological function, Biochem. J. 300 (1994) 281–290. [13] R. Atreya, J. Mudter, S. Finotto, J. Müllberg, T. Jostock, S. Wirtz, M. Schütz, B. Bartsch, M. Holtmann, C. Becker, D. Strand, J. Czaja, J.F. Schlaak, H.A. Lehr, F. Autschbach, G. Schürmann, N. Nishimoto, K. Yoshizaki, H. Ito, T. Kishimoto, P.R. Galle, S. Rose-John, M.F. Neurath, Blockade of IL-6 transsignaling abrogates established experimental colitis in mice by suppression of the antiapoptotic resistance of lamina propria T cells, Nat. Med. 6 (2000) 583–588. [14] C. Becker, M.C. Fantini, C. Schramm, H.A. Lehr, S. Wirtz, A. Nikolaev, J. Burg, S. Strand, R. Kiesslich, S. Huber, H. Ito, N. Nishimoto, K. Yoshizaki, T. Kishimoto, P.R. Galle, M. Blessing, S. Rose-John, M.F. Neurath, TGF-beta suppresses tumor progression in colon cancer by inhibition of IL-6 trans-signaling, Immunity 21 (2004) 491–501. [15] A. Chalaris, B. Rabe, K. Paliga, H. Lange, T. Laskay, C.A. Fielding, S.A. Jones, S. RoseJohn, J. Scheller, Apoptosis is a natural stimulus of IL6R shedding and contributes to the pro-inflammatory trans-signaling function of neutrophils, Blood 110 (2007) 1748–1755. [16] B. Rabe, A. Chalaris, U. May, G.H. Waetzig, D. Seegert, A.S. Williams, S.A. Jones, S. Rose-John, J. Scheller, Transgenic blockade of interleukin 6 transsignaling abrogates inflammation, Blood 111 (2008) 1021–1028. [17] J. Scheller, J. Grötzinger, S. Rose-John, Updating IL-6 classic- and trans-signaling, Signal Transduct. 6 (2006) 240–259. [18] S. Grivennikov, M. Karin, Autocrine IL-6 signaling: a key event in tumorigenesis? Cancer Cell 13 (2008) 7–9. [19] S. Rose-John, G.H. Waetzig, J. Scheller, J. Grotzinger, D. Seegert, The IL-6/sIL-6R complex as a novel target for therapeutic approaches, Expert Opin. Ther. Targets 11 (2007) 613–624. [20] M.S. Brown, J. Ye, R.B. Rawson, J.L. Goldstein, Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans, Cell 100 (2000) 391–398. [21] C. Haass, Take five-BACE and the gamma-secretase quartet conduct Alzheimer's amyloid beta-peptide generation, EMBO J. 23 (2004) 483–488. [22] M.S. Wolfe, W. Xia, C.L. Moore, D.D. Leatherwood, B. Ostaszewski, T. Rahmati, I.O. Donkor, D.J. Selkoe, Peptidomimetic probes and molecular modeling suggest that

A. Chalaris et al. / Biochimica et Biophysica Acta 1803 (2010) 234–245

[23] [24] [25]

[26]

[27]

[28]

[29]

[30] [31]

[32]

[33]

[34]

[35]

[36]

Alzheimer's gamma-secretase is an intramembrane-cleaving aspartyl protease, Biochemistry 38 (1999) 4720–4727. H. Steiner, R. Fluhrer, C. Haass, Intramembrane proteolysis by gamma-secretase, J. Biol. Chem. 283 (2008) 29627–29631. J.S. Mumm, R. Kopan, Notch signaling: from the outside in, Dev. Biol. 228 (2000) 151–165. M. Bentahir, O. Nyabi, J. Verhamme, A. Tolia, K. Horré, J. Wiltfang, H. Esselmann, B. De Strooper, Presenilin clinical mutations can affect gamma-secretase activity by different mechanisms, J. Neurochem. 96 (2006) 732–742. S. Shah, S.F. Lee, K. Tabuchi, Y.H. Hao, C. Yu, Q. LaPlant, H. Ball, C.E. Dann 3rd, T. Sudhof, G. Yu, Nicastrin functions as a gamma-secretase-substrate receptor, Cell 122 (2005) 435–447. L. Chávez-Gutiérrez, A. Tolia, E. Maes, T. Li, P.C. Wong, B. de Strooper, Glu(332) in the Nicastrin ectodomain is essential for gamma-secretase complex maturation but not for its activity, J. Biol. Chem. 283 (2008) 20096–20105. C. Sato, Y. Morohashi, T. Tomita, T. Iwatsubo, Structure of the catalytic pore of gamma-secretase probed by the accessibility of substituted cysteines, J. Neurosci. 26 (2006) 12081–12088. A. Tolia, L. Chávez-Gutiérrez, B. De Strooper, Contribution of presenilin transmembrane domains 6 and 7 to a water-containing cavity in the gammasecretase complex, J. Biol. Chem. 281 (2006) 27633–27642. R. Kopan, M.X. Ilagan, Gamma-secretase: proteasome of the membrane? Nat. Rev. Mol. Cell Biol. 5 (2004) 499–504. B. Schuster, M. Kovaleva, Y. Sun, P. Regenhard, V. Matthews, J. Grötzinger, S. RoseJohn, K.-J. Kallen, Signalling of human CNTF revisited: the interleukin-6 (IL-6) receptor can serve as an a-receptor for ciliary neurotrophic factor (CNTF), J. Biol. Chem. 278 (2003) 9528–9535. C. Hundhausen, D. Misztela, T.A. Berkhout, N. Broadway, P. Saftig, K. Reiss, D. Hartmann, F. Fahrenholz, R. Postina, V. Matthews, K.J. Kallen, S. Rose-John, A. Ludwig, The disintegrin-like metalloproteinase ADAM10 is involved in constitutive cleavage of CX3CL1 (fractalkine) and regulates CX3CL1-mediated cell–cell adhesion, Blood 102 (2003) 1186–1195. R. Ketteler, S. Glaser, O. Sandra, U.M. Martens, U. Klingmuller, Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors, Gene Ther. 9 (2002) 477–487. A. Herreman, D. Hartmann, W. Annaert, P. Saftig, K. Craessaerts, L. Serneels, L. Umans, V. Schrijvers, F. Checler, H. Vanderstichele, V. Baekelandt, R. Dressel, P. Cupers, D. Huylebroeck, A. Zwijsen, F. Van Leuven, B. De Strooper, Presenilin 2 deficiency causes a mild pulmonary phenotype and no changes in amyloid precursor protein processing but enhances the embryonic lethal phenotype of presenilin 1 deficiency, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 11872–11877. C.J. McMahan, J.L. Slack, B. Mosley, D. Cosman, S.D. Lupton, L.L. Brunton, C.E. Grubin, J. M. Wignall, N.A. Jenkins, C.I. Brannan, N.G. Copeland, K. Huebner, C.M. Croce, L.A. Cannizzarro, D. Benjamin, S.K. Dower, M.K. Spriggs, J.E. Sims, A novel IL-1 receptor, cloned from B cells by mammalian expression, is expressed in many cell types, EMBO J. 10 (1991) 2821–2832. A. Weidemann, S. Eggert, F.B. Reinhard, M. Vogel, K. Paliga, G. Baier, C.L. Masters, K. Beyreuther, G. Evin, A novel epsilon-cleavage within the transmembrane domain

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44] [45] [46] [47] [48] [49]

[50]

[51]

245

of the Alzheimer amyloid precursor protein demonstrates homology with Notch processing, Biochemistry 41 (2002) 2825–2835. S. Eggert, K. Paliga, P. Soba, G. Evin, C.L. Masters, A. Weidemann, K. Beyreuther, The proteolytic processing of the amyloid precursor protein gene family members APLP-1 and APLP-2 involves alpha-, beta-, gamma-, and epsilon-like cleavages: modulation of APLP-1 processing by n-glycosylation, J. Biol. Chem. 279 (2004) 18146–18156. A. Schulte, B. Schulz, M.G. Andrzejewski, C. Hundhausen, S. Mletzko, J. Achilles, K. Reiss, K. Paliga, C. Weber, S. Rose-John, A. Ludwig, Sequential processing of the transmembrane chemokines CX3CL1 and CXCL16 by α- and γ-secretases, Biochem. Biophys. Res. Commun. 358 (2007) 233–240. C. Kaether, S. Schmitt, M. Willem, C. Haass, Amyloid precursor protein and Notch intracellular domains are generated after transport of their precursors to the cell surface, Traffic 7 (2006) 408–415. C. Hundhausen, A. Schulte, B. Schulz, M.G. Andrzejewski, N. Schwarz, P. von Hundelshausen, U. Winter, K. Paliga, K. Reiss, P. Saftig, C. Weber, A. Ludwig, Regulated shedding of transmembrane chemokines by the disintegrin and metalloproteinase 10 facilitates detachment of adherent leukocytes, J. Immunol. 178 (2007) 8064–8072. Y. Morohashi, T. Kan, Y. Tominari, H. Fuwa, Y. Okamura, N. Watanabe, C. Sato, H. Natsugari, T. Fukuyama, T. Iwatsubo, T. Tomita, C-terminal fragment of presenilin is the molecular target of a dipeptidic gamma-secretase-specific inhibitor DAPT (N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, J. Biol. Chem. 281 (2006) 14670–14676. S.S. Huppert, A. Le, E.H. Schroeter, J.S. Mumm, M.T. Saxena, L.A. Milner, R. Kopan, Embryonic lethality in mice homozygous for a processing-deficient allele of Notch1, Nature 405 (2000) 966–970. K. Wilhelmsen, P. van der Geer, Phorbol 12-myristate 13-acetate-induced release of the colony-stimulating factor 1 receptor cytoplasmic domain into the cytosol involves two separate cleavage events, Mol. Cell. Biol. 24 (2004) 454–464. S. Meiners, A. Ludwig, V. Stangl, K. Stangl, Proteasome inhibitors: poisons and remedies, Med. Res. Rev. 28 (2008) 309–327. S. Dimitrov, T. Lange, C. Benedict, J. Scheller, S. Rose-John, S. Jones, J. Born, Sleep enhances IL-6 trans-signaling in humans, FASEB J. 20 (2006) 2174–2176. G. Murphy, A. Murthy, R. Khokha, Clipping, shedding and RIPping keep immunity on cue, Trends Immunol. 29 (2008) 75–82. N. Landman, T.W. Kim, Got RIP? Presenilin-dependent intramembrane proteolysis in growth factor receptor signaling, Cytokine Growth Factor Rev 15 (2004) 337–351. S.J. Bray, Notch signalling: a simple pathway becomes complex, Nat. Rev. Mol. Cell Biol. 7 (2006) 678–689. D. Nanba, A. Mammoto, K. Hashimoto, S. Higashiyama, Proteolytic release of the carboxy-terminal fragment of proHB-EGF causes nuclear export of PLZF, J. Cell Biol. 163 (2003) 489–502. M. Hieda, M. Isokane, M. Koizumi, C. Higashi, T. Tachibana, M. Shudou, T. Taguchi, Y. Hieda, S. Higashiyama, Membrane-anchored growth factor, HB-EGF, on the cell surface targeted to the inner nuclear membrane, J. Cell Biol. 180 (2008) 763–769. A.S. Martens, J.G. Bode, P.C. Heinrich, L. Graeve, The cytoplasmic domain of the interleukin-6 receptor gp80 mediates its basolateral sorting in polarized Madin– Darby canine kidney cells, J. Cell Sci. 113 (2000) 3593-3560.